Anti-CCR5 antibody

ABSTRACT

The invention is directed an anti-CCR5 antibody which comprises (i) two light chains, each light chain comprising the expression product of a plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavy chains, each heavy chain comprising an expression product of either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099) or a fragment thereof which binds to CCR5 on the surface of a human cell.

[0001] This application is a continuation-in-part of and claims thepriority of U.S. Provisional Application No. 60/358,886, filed Feb. 22,2002, the contents of which are hereby incorporated by reference intothis application.

[0002] Throughout this application, various publications are referencedby Arabic numerals. Full citations for these publications may be foundat the end of the specification immediately preceding the claims. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

[0003] Human immunodeficiency virus type 1 (HIV-1) induces viral-to-cellmembrane fusion to gain entry into target cells (8, 15, 66). The firsthigh-affinity interaction between the virion and the cell surface is thebinding of the viral surface glycoprotein gp120 to the CD4 antigen (13,30, 41, 42). This in turn induces conformational changes in gp120, whichenable it to interact with one of several chemokine receptors (4, 5, 21,36). The CC-chemokine receptor CCR5 is the major co-receptor formacrophage-tropic (R5) strains, and plays a crucial role in the sexualtransmission of HIV-1 (4, 5, 21, 36). T cell line-tropic (X4) virusesuse CXCR4 to enter target cells, and usually, but not always, emergelate in disease progression or as a consequence of virus propagation intissue culture (4, 5, 21, 36). Some primary HIV-1 isolates aredual-tropic (R5×4) since they can use both co-receptors, though notalways with the same efficiency (11, 57). Mutagenesis studies coupledwith the resolution of the gp120 core crystal structure demonstratedthat the co-receptor-binding site on gp120 comprises several conservedresidues (32, 53, 65).

[0004] It has been demonstrated that tyrosines and negatively chargedresidues in the amino-terminal domain (Nt) of CCR5 are essential forgp120 binding to the co-receptor, and for HIV-1 fusion and entry (6, 18,20, 22, 28, 31, 52, 54). Residues in the extracellular loops (ECL) 1-3of CCR5 were dispensable for co-receptor function, yet the CCR5inter-domain configuration had to be maintained for optimal viral fusionand entry (24) This led to the conclusion either that gp120 formsinteractions with a diffuse surface on the ECLs, or that the Nt ismaintained in a functional conformation by bonds with residues in theECLs. Studies with chimeric co-receptors and anti-CCR5 monoclonalantibodies have also shown the importance of the extracellular loops forviral entry (5, 54, 64).

[0005] Molecules that specifically bind to CCR5 and CXCR4 and blockinteractions with their ligands are a powerful tool to further probe thestructure/function relationships of the co-receptors. Characterizingsuch compounds could also assist in designing effective therapeuticagents that target co-receptor-mediated steps of viral entry. Inhibitorsof CCR5 or CXCR4 co-receptor function identified to date are diverse innature and include small molecules, peptides, chemokines and theirderivatives, and monoclonal antibodies (mAbs). The mechanisms of actionof the small molecules that block entry by interfering with CXCR4co-receptor function are not well understood (17, 49, 55, 68). One suchinhibitor, the anionic small molecule AMD3100, depends on residues inECL2 and the fourth trans-membrane (TM) domain of CXCR4 to inhibit viralentry, but it is not clear whether it does so by disrupting gp120binding to CXCR4 or post-binding steps leading to membrane fusion (16,34, 55) To date, no small molecules have been reported that specificallyblock CCR5-mediated HIV-1 entry. Inhibition of HIV-1 entry by chemokinesis mediated by at least two distinct mechanisms: blockage of thegp120/co-receptor interaction and internalization of thechemokine/receptor complex (3, 26, 59, 63). The variant AOP-RANTES alsoinhibits recycling of CCR5 to the cell surface (40, 56). Variants suchas RANTES 9-68 and Met-RANTES only prevent the gp120/CCR5 interactionand do not down-regulate CCR5 (67). SDF-1 variants presumably actthrough a similar mechanism to block viral entry mediated by CXCR4 (12,27, 39). Only one anti-CXCR4 mAb, 12G5, has been characterized for itsanti-viral properties. The efficiency of 12G5 inhibition of viral entryhas been reported to be both cell- and isolate-dependent (43, 58). ThismAb binds to the ECL2 of CXCR4, but the mechanism by which it inhibitsentry is unknown (7). Few of the anti-CCR5 mAbs characterized to dateefficiently prevent HIV-1 entry (28, 64). Interestingly, mAbs whoseepitopes lie in the Nt domain of CCR5, which contains the gp120-bindingsite, inhibit viral fusion and entry less efficiently than mAb 2D7,whose epitope lies in ECL2. 2D7 also antagonizes CC-chemokine activity(64).

[0006] A panel of six murine mAbs, designated PA8, PA9, PA10, PA11, PA12and PA14 have been isolated and characterized. All six mAbs specificallybound to CCR⁵⁺ cells but with different efficiencies that were celltype-dependent. Epitope mapping studies identified the residues that areimportant for mAb binding and also revealed information about thefolding and interactions of the CCR5 extracellular domains. All mAbsinhibited HIV-1 fusion and entry, but there was no correlation betweenthe ability of a mAb to inhibit fusion and entry and its ability toinhibit binding of gp120/sCD4 to CCR5⁺ cells.

SUMMARY OF THE INVENTION

[0007] This invention provides an anti-CCR5 antibody which comprises (i)two light chains, each light chain comprising the expression product ofa plasmid designated pVK:HuPRO140-VK (ATCC Deposit DesignationPTA-4097), and (ii) two heavy chains, each heavy chain comprising theexpression product of either a plasmid designated pVg1:HuPRO140 HG2-VH(ATCC Deposit Designation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), or a fragment ofsuch antibody, which binds to CCR5 on the surface of a human cell.

[0008] This invention also provides an anti-CCR5 antibody comprising twolight chains, each chain comprising consecutive amino acids, the aminoacid sequence of which is set forth in SEQ ID NO: 6, and two heavychains, each heavy chain comprising consecutive amino acids, the aminoacid sequence of which is set forth in SEQ ID NO: 9.

[0009] This invention also provides an anti-CCR5 antibody comprising twolight chains, each chain comprising consecutive amino acids, the aminoacid sequence of which is set forth in SEQ ID NO: 6, and two heavychains, each heavy chain comprising consecutive amino acids, the aminoacid sequence of which is set forth in SEQ ID NO: 12.

[0010] This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO: 6. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO: 5.

[0011] This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO: 9. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO: 8.

[0012] This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO: 12. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO: 11.

[0013] This invention also provides a composition comprising at leastone anti-CCR5 antibody, or a fragment thereof, as described above,together with a carrier.

[0014] This invention also provides a composition comprising theanti-CCR5 antibody, or a fragment thereof, having attached thereto amaterial such as a radioisotope, a toxin, polyethylene glycol, acytotoxic agent and/or a detectable label.

[0015] This invention also provides a method of inhibiting infection ofa CD4+ cell which comprises contacting the CD4+ cell with an antibodywhich comprises (i) two light chains, each light chain comprising theexpression product of a plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097), and (ii) two heavy chains, each heavy chaincomprising the expression product of either a plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099), or a fragment of such antibody which binds to CCR5 on thesurface of a CD4+ cell, in an amount and under conditions such thatfusion of HIV-1 or an HIV-1-infected cell to the CD4+ cell is inhibited,thereby inhibiting HIV-1 infection of the CD4+ cell.

[0016] This invention also provides a method of treating a subjectafflicted with HIV-1 which comprises administering to the subject aneffective HIV-1 treating dosage of an anti-CCR5 antibody comprising (i)two light chains, each light chain comprising the expression product ofa plasmid designated pVK:HuPRO140-VK (ATCC Deposit DesignationPTA-4097), and (ii) two heavy chains, each heavy chain comprising theexpression product of either a plasmid designated pVg1:HuPRO140 HG2-VH(ATCC Deposit Designation PTA-4098) or a plasmid designatedpVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), or afragment of such antibody, which binds to CCR5 on the surface of a humancell, under conditions effective to treat the HIV-1-infected subject.

[0017] This invention also provides a method of preventing a subjectfrom contracting an HIV-1 infection which comprises administering to thesubject an effective HIV-1 infection-preventing dosage amount of ananti-CCR5 antibody comprising (i) two light chains, each light chaincomprising the expression product of a plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavychains, each heavy chain comprising the expression product of either aplasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCCDeposit Designation PTA-4099), or a fragment of such antibody, whichbinds to CCR5 on the surface of a human cell, under conditions effectiveto prevent the HIV-1, infection in the subject.

[0018] This invention also provides an anti-CCR5 antibody conjugatecomprising an anti-CCR5 antibody which comprises (i) two light chains,each light chain comprising the expression product of a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii)two heavy chains, each heavy chain comprising the expression product ofeither a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), or a fragment of suchantibody which binds to CCR5 on the surface of a human cell, conjugatedto at least one polymer.

[0019] This invention also provides a method of inhibiting infection ofa CCR5+ cell by HIV-1 comprising administering to a subject at risk ofHIV-1 infection the above-described conjugate in an amount and underconditions effective to inhibit infection of CCR5+ cells of the subjectby HIV-1.

[0020] This invention also provides a method of treating an HIV-1infection in a subject comprising administering the above-describedconjugate to an HIV-1-infected subject in an amount and under conditionseffective to treat the subject's HIV-1 infection.

[0021] This invention also provides a transformed host cell comprisingat least two vectors, at least one vector comprising a nucleic acidsequence encoding heavy chains of an anti-CCR5 antibody, and at leastone vector comprising a nucleic acid sequence encoding light chains ofthe anti-CCR5 antibody, wherein the anti-CCR5 antibody comprises twoheavy chains having the amino acid sequence set forth in SEQ ID NO: 9,and two light chains having the amino acid sequence set forth in SEQ IDNO: 6.

[0022] This invention also provides a transformed host cell comprisingat least two vectors, at least one vector comprising a nucleic acidsequence encoding heavy chains of, an anti-CCR5 antibody, and at leastone vector comprising a nucleic acid sequence encoding light chains ofthe anti-CCR5 antibody, wherein the anti-CCR5 antibody comprises twoheavy chains having the amino acid sequence set forth in SEQ ID NO: 12and two light chains having the amino acid sequence set forth in SEQ IDNO: 6.

[0023] This invention also provides a vector comprising a nucleic acidsequence encoding a heavy chain of an anti-CCR5 antibody, wherein theheavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0024] This invention also provides a vector comprising a nucleic acidsequence encoding a heavy chain of an anti-CCR5 antibody, wherein theheavy chain comprises the amino acid sequence set forth in SEQ ID NO:12.

[0025] This invention also provides a process for producing an anti-CCR5antibody which comprises culturing a host cell containing therein (i) aplasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097),and (ii) either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099) under conditionspermitting the production of an antibody comprising two light chainsencoded by the plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097) and two heavy chains encoded either by the plasmiddesignated pVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) orby the plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC DepositDesignation PTA-4099), so as to thereby produce an anti-CCR5 antibody.

[0026] This invention also provides a process for producing an anti-CCR5antibody which comprises a)transforming a host cell with (i) a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097)and (ii)either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), and b) culturing thetransformed host cell under conditions permitting production of anantibody comprising two light chains encoded by the plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097) and two heavy chainsencoded either by the plasmid designated pVg1:HuPRO140 HG2-VH (ATCCDeposit Designation PTA-4098) or by the plasmid designated pVg1HuPRO140(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), so as to therebyproduce an anti-CCR5 antibody.

[0027] This invention also provides a kit for use in a process ofproducing an anti-CCR5 antibody. The kit comprises a) a vectorcomprising a nucleic acid sequence encoding a light chain of ananti-CCR5 antibody, wherein the light chain comprises the amino acidsequence set forth in SEQ ID NO: 6, and b) a vector comprising a nucleicacid sequence encoding a heavy chain of an anti-CCR5 antibody, whereinthe heavy chain comprises the amino acid sequence set forth in SEQ IDNO: 9, or a vector comprising a nucleic acid sequence encoding a heavychain of an anti-CCR5 antibody, wherein the heavy chain comprises theamino acid sequence set forth in SEQ ID NO: 12.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1

[0029] Binding of Anti-CCR5 Monoclonal-Antibodies to CCR5⁺ Cells

[0030] Flow cytometry was used to detect CCR5 protein expression on thesurface of L1.2-CCR5⁺ cells and freshly isolated, PHA/IL-2-stimulatedPEMC. Cells were incubated with saturating concentrations of each mAb,which were detected with a PE-labeled anti-mouse IgG reporter antibody.Results from a representative experiment are shown. Results for each mAbare expressed both in mean fluorescence intensities (m.f.i.) and in %gated cells. Since PA8-PA12 and PA14 are all of the IgG1 subclass, theirm.f.i. are directly comparable. 2D7 is an IgG2a.

[0031]FIG. 2

[0032] CI Values for Different Combinations of mAbs and Viral Inhibitors

[0033] Experiments like those described in the legend of FIG. 7 wereperformed for different combinations of viral entry inhibitors.Anti-CCR5 mAbs were tested in combination with each other,CC-chemokines, and CD4-IgG2, which inhibits HIV-1 attachment to targetcells. The PA11 and PA12 concentration range was 0-250 μg/ml; the 2D7and PA14 concentration range was 0-25 μg/ml; the RANTES concentrationrange was 0-250 ng/ml; the CD4-IgG2 concentration range was 0-25 μg/ml.The concentrations of single-agents or their mixtures required toproduce 50% and 90% inhibition of fusion or entry were quantitativelycompared in a term known as the Combination Index (CI).

[0034]FIG. 3

[0035] IC₅₀ Values for Inhibition of Cell-Cell Fusion, Viral Entry andgp120 /sCD4 Binding by Anti-CCR5 mAbs

[0036] For comparative purposes we have summarized the IC₅₀ valuesobtained in the different assays that the anti-CCR5 mAbs were tested in.IC₅₀ values were only calculated for mAbs that could inhibit >90% offusion, entry or binding.

[0037]FIG. 4

[0038] Epitope Mapping of Anti-CCR5 mAbs:

[0039] A two color staining protocol was used to assess binding of mAbsto mutant CCR5 proteins, tagged at the C-terminus with the HA peptide.HeLa cells expressing CCR5 point mutants were incubated with saturatingconcentrations of each mAb followed by detection with a PE-labeledanti-mouse IgG. Cell surface co-receptor expression was measured bydouble-staining of the cells with a FITC labeled anti-HA mAb. The fourgrids correspond to the four extracellular domains of CCR5. The firstrow of every grid indicates the amino acid sequence of the correspondingCCR5 extracellular domain (SEQ ID NOS: 1-4). Binding of anti-CCR5 mAbsto the alanine mutant of each residue is expressed as a percentage ofbinding to wild-type CCR5, as described in Materials and Methods.

[0040]FIG. 5

[0041] Inhibition of Calcium Mobilization into CCR5⁺ cells by Anti-CCR5mAbs

[0042] L1.2-CCR5⁺ cells were loaded with Indo-1AM and stimulatedsequentially with an anti-CCR5 mAb or PBS, followed with RANTES (a).Fluorescence changes were measured with a spectrofluorometer and thetracings are from a representative experiment. Calcium flux inhibitionby PA14 and 2D7 was tested for a wide range of mAb concentrations (b).Results are plotted as % inhibition of calcium influx=[1−(relativefluorescence in the presence of mAb÷relative fluorescence in the absenceof mAb)]×100%, and are means of values from three independentexperiments.

[0043]FIG. 6

[0044] Inhibition of CCR5 Co-Receptor Function by Anti-CCR5 mAbs

[0045] Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested inthe RET assay (a). 0-250 μg/ml of PA8-PA12, or 0-25 μg/ml of PA14 or2D7, were added to a mix of HeLa-Env_(JR-FL) ⁺ and PM1 cells, labeledwith F18 and R18 respectively. Fluorescence RET was measured after 4 hof incubation. Results are mean values from three independentexperiments and are expressed as % inhibition of fusion=[1−(% RET in thepresence of mAb÷% RET in the absence of mAb)]×100%. Inhibition of HIV-1entry by anti-CCR5 mAbs was tested in a single round of replicationluciferase based entry assay (b). U87-CD4⁺CCR5⁺ cells were infected withNLluc⁺env⁻ reporter virus carrying the JR-FL envelope in the presence of0-250 μg/ml of PA8-PA12, or 0-25 μg/ml PA14 or 2D7. Luciferase activity(relative light units, r.l.u.) was measured in cell lysates 72 hpost-infection. Results are from a representative experiment and areexpressed as % inhibition of entry=[1−(r.l.u. in the presence ofmAb÷r.l.u. in the absence of mAb)]×100%. Binding of biotinylated[b]gp120, sCD4 and b-gp120-CD4 complexes to L1.2-CCR5⁺ cells (c). Strongbinding is observed when gp120 derived from the R5 virus HIV-1_(JR-FL)is complexed with an equimolar amount of sCD4. No binding is observed inthe absence of sCD4 or. for gp120 derived from the X4 virus HIV-1_(LAI).Background binding to CCR5-L1.2 cells has been subtracted from allcurves. Inhibition of gp120/sCD4 binding to L1.2-CCR5⁺ cells was testedin the presence of varying concentrations of each antibody (d). Cellswere pre-incubated in 96-well plates with an anti-CCR5 mAb followed byan incubation with a saturating concentration of biotinylatedgp120/sCD4. Finally, binding of PE-labeled streptavidin to cells wasmeasured using a fluorescence plate reader. Results are from arepresentative experiment and are expressed as % inhibition ofgp120/sCD4 binding=[1−(m.f.i. in the presence of mAb÷m.f.i. in theabsence of mAb)]×100%.

[0046]FIG. 7

[0047] Synergistic Inhibition of Cell-Cell Fusion by PA12 and 2D7

[0048] Dose-response curves were obtained for the mAbs used individuallyand in combination. 0-50 μg/ml of PA12, 0-25 μg/ml 2D7, or a combinationof the two in a 2:1 ratio, were added to a mix of HeLa-Env_(JR-FL) ⁺ andPM1 cells, labeled with R18 and F18 respectively. Fluorescence RET wasmeasured after 4 hours of incubation. Results are expressed as %inhibition of fusion and are the means of values from three independentexperiments. Data were analyzed using the median effect principle, whichcan be written

f=1/[1+(K/c) ^(m)]  (1)

[0049] where f is the fraction affected/inhibited, c is concentration, Kis the concentration of agent required to produce the median effect, andm is an empirical coefficient describing the shape of the dose-responsecurve. Equation (1) is a generalized form of the equations describingMichaelis-Menton enzyme kinetics, Langmuir adsorption isotherms, andHenderson-Hasselbalch ionization equilibria, for which m=1. In thepresent case, K is equal to the IC₅₀ value. K and m were determined bycurve-fitting the dose-response curves and Equation (1) was rearrangedto allow calculation of c for a given f. The best-fit parameters for Kand c are 8.8 μg/ml and 0.54 for PA12, 0.36 μg/ml and 0.68 for 2D7, and0.11 μg/ml and 1.1 for their combination. These curves are plotted andindicate a reasonable goodness-of-fit between experiment and theory.

[0050]FIG. 8

[0051] This figure shows the amino acid sequence of the light chainvariable region of a humanized version of mouse anti-CCR5 antibody PA14(SEQ ID NO: 6) and the nucleic acid sequence encoding the same (SEQ IDNO: 5), in accordance with the invention. SEQ ID NO: 7 identifies theregion of SEQ ID NO: 5 which codes for the amino acid sequence set forthin SEQ ID NO: 6. This light chain variable region is present in theantibodies designated herein as PRO 140 #1 and #2. Thecomplementarity-determining regions (“CDRs”) are underlined.

[0052]FIG. 9

[0053] This figure shows the amino acid sequence of a first heavy chainvariable region of a humanized version of mouse anti-CCR5 antibody PA14(SEQ ID NO: 9), and the nucleic acid sequence encoding the same (SEQ IDNO: 8), in accordance with the invention. SEQ ID NO: 10 identifies theregion of SEQ ID NO: 8 that codes for the amino acid sequence set forthin SEQ ID NO: 9. This heavy chain variable region is present in theantibody designated herein as PRO 140 #2. The CDRs are underlined.

[0054]FIG. 10

[0055] This figure shows the amino acid sequence of a second heavy chainvariable region of a humanized version of mouse humanized anti-CCR5antibody PA14 (SEQ ID NO: 12) and the nucleic acid sequence encoding thesame (SEQ ID NO: 11) in accordance with the invention. SEQ ID NO: 13identifies the region of SEQ ID NO: 11 that codes for the amino acidsequence set forth in SEQ ID NO: 12. This heavy chain variable region ispresent in the antibody designated herein as PRO 140 #1. The CDRs areunderlined.

[0056]FIG. 11

[0057] Single-Dose of Humanized CCR5 Antibody Potently Reduces ViralLoads in vivo

[0058] SCID mice were reconstituted with normal human PBMC and infectedwith HIV-1_(JR-CSF). When a viral steady state was reached, the animalswere treated with a single 1 milligram i.p. dose of humanized CCR5antibody (PRO 140) or isotype control antibody and monitored for plasmaHIV RNA (Roche Amplicor Assay).

[0059]FIG. 12

[0060] Sustained Reduction in Viral Load

[0061] SCID mice were reconstituted with normal human PBMC and infectedwith HIV-1_(JR-CSF). When a viral steady state was reached, the animalswere treated i.p. with 0.1 mg doses of humanized CCR5 antibody (PRO140)every three days and monitored for plasma HIV RNA (Roche AmplicorAssay).

[0062]FIG. 13

[0063] Demonstrates that there was no depletion of lymphocytes with theuse of the CCR5 antibody (PRO 140) prepared in accordance with theinvention.

[0064]FIG. 14

[0065] Humanized CCR5 Antibody (PRO140) Potently Blocks CCR5-mediatedHIV-1 Cell-Cell Fusion.

[0066] Murine CCR5 antibody was humanized using the method ofcomplementarity-determining region (CDR) grafting and frameworksubstitutions. Humanized CCR5 antibodies (PRO 140 #1 and PRO 140 #2)were expressed in Sp2/0 cells, purified by protein A chromatography andtested for the ability to block replication of HIV-1_(JR-FL)env-mediated membrane fusion as described (Litwin, et al., J, Virol.,70:6437, 1996).

[0067]FIG. 15

[0068] Humanized CCR5 Antibody (PRO 140) Mediates Potent,Subtype-Independent Inhibition of HIV-1.

[0069] CCR5 Antibodies (Pro 140 #1 and #2) according to the inventionwere tested for the ability to block replication of wild-type HIV-1 inperipheral blood mononuclear cells (PBMCs) as described (Trkola et al.,J. Virol., 72:396, 1998). The extent of viral replication was measuredby assaying the p24 antigen content of 7-day PBMC culture supernatants.

[0070]FIG. 16

[0071] This figure provides a map of plasmid pVK-HuPRO140 encoding thelight plasmid chain variable region shown in FIG. 8 as well as the humanKappa constant regions as described in Co et al., J.Immunol., 148:1149,1992.

[0072]FIG. 17

[0073] This figure provides a map of plasmid pVg4-HuPRO140 HG2 encodingthe heavy chain variable region shown in FIG. 9 as well as the humanheavy chain constant regions, CH1, hinge, CH2, and CH3, of human IgG4 asdescribed in Co et al, Supra.

[0074]FIG. 18

[0075] This figure provides a map of plasmid pVg4-HuPRO140 (mut B+D+I)encoding the heavy chain variable region shown in FIG. 10 as well as thehuman heavy chain constant regions, CH1, hinge, CH2, and CH3, of humanIgG4 as described in Co et al, Supra.

[0076]FIG. 19

[0077] Hu PRO140 Blocks HIV-1 But Not RANTES Signaling

[0078] PRO140 antibodies according to the invention were tested for theability to block RANTES-induced calcium mobilization in L1.2-CCR5 cells(Olson, et al., J. Virol., 72:396, 1998). This figure shows that ahumanized CCR5 antibody (huPRO140) blocks HIV-1 but not RANTESsignaling.

DETAILED DESCRIPTION OF THE INVENTION

[0079] The plasmids designated as HuPRO140-VK, HuPRO140 (mut+B+D+I)-VH,and HuPRO140 HG2-VH, which are referred to in FIGS. 16, 18, and 17 aspVK-HuPRO140, pVg4-HuPRO140 (mut B+D+I) and pVg4-HuPRO140 HG2,respectively, were deposited with the American Type Culture Collection,Manassas, Va., U.S.A. 20108 on Feb. 22, 2002, under ATCC Accession Nos.PTA 4097, PTA 4099 and PTA 4098 respectively. These deposits were madepursuant to the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure (Budapest Treaty).

[0080] This invention provides a composition for inhibiting HIV-1infection comprising at least two compounds in synergistically effectiveamounts for inhibiting HIV-1 infection, wherein at least one of thecompounds prevents with the productive interaction between HIV-1 and anHIV-1 fusion co-receptor.

[0081] As used herein, “composition” means a mixture. The compositionsinclude but are not limited to those suitable for oral, rectal,intravaginal, topical, nasal, opthalmic, or parenteral administration toa subject. As used herein, “parenteral” includes but is not limited tosubcutaneous, intravenous, intramuscular, or intrasternal injections orinfusion techniques.

[0082] As used herein, “HIV-1” means the human immunodeficiency virustype-1. HIV-1 includes but is not limited to extracellular virusparticles and the forms of HIV-1 found in HIV-1 infected cells.

[0083] As used herein, “HIV-1 infection” means the introduction of HIV-1genetic information into a target cell, such as by fusion of the targetcell membrane with HIV-1 or an HIV-1 envelope glycoprotein⁺ cell. Thetarget cell may be a bodily cell of a subject. In the preferredembodiment, the target cell is a bodily cell from a human subject.

[0084] As used herein, “inhibiting HIV-1 infection” means the reductionof the amount of HIV-1 genetic information introduced into a target cellpopulation as compared to the amount that would be introduced withoutsaid composition.

[0085] As used herein, “compound” means a molecular entity, includingbut not limited to peptides, polypeptides, and other organic orinorganic molecules and combinations thereof.

[0086] As used herein, “synergistically effective” means that thecombined effect of the compounds when used in combination is greaterthan their additive effects when used individually.

[0087] As used herein, “productive interaction” means that theinteraction of HIV-1 and the HIV-1 co-receptor would lead to the fusionof said HIV-1 or HIV-1 envelope glycoprotein⁺ cell and the membranebearing the co-receptor.

[0088] As used herein, “prevents the productive interaction” means thatthe amount of interaction is reduced as compared to the amount thatwould occur without the compound. The interactions may be prevented bymasking or altering interactive regions on the co-receptor or HIV-1 orby altering the expression, aggregation, conformation, or associationstate of the co-receptor.

[0089] As used herein, “HIV-1 fusion co-receptor” means a cellularreceptor that mediates fusion between the target cell expressing thereceptor and HIV-1 or an HIV-1 -envelope glycoprotein⁺ cell. HIV-1fusion co-receptors include but are not limited to CCR5, CXCR4 and otherchemokine receptors.

[0090] This invention also provides a composition which inhibits fusionof HIV-1 or an HIV-1 envelope glycoprotein⁺ cell to a target cell,comprising at least two compounds in synergistically effective amountsfor inhibiting fusion of HIV-1 or, an HIV-1 envelope glycoprotein⁺ cellto a target cell, wherein at least one of the compounds prevents theproductive interaction between HIV-1 and an HIV-1 fusion co-receptor.

[0091] As used herein, “fusion” means the joining or union of the lipidbilayer membranes found on mammalian cells or viruses such as HIV-1.This process is distinguished from the attachment of HIV-1 to a targetcell. Attachment is mediated by the binding of the HIV-1 exteriorglycoprotein to the human CD4 receptor, which is not a fusionco-receptor.

[0092] As used herein, “inhibits” means that the amount is reduced ascompared with the amount that would occur without the composition.

[0093] As used herein, “target cell” means a cell capable of beinginfected by or fusing with HIV-1 or HIV-1 infected cells.

[0094] As used herein, “chemokine” means a cytokine that can stimulateleukocyte movement. They may be characterized as either cys-cys orcys-X-cys depending on whether the two amino terminal cysteine residuesare immediately adjacent or separated by one amino acid. It includes butis not limited to RANTES, MIP-1α, MIP-1β, SDF-1 or another chemokinewhich blocks HIV-1 infection.

[0095] In one embodiment of the above compositions, the co-receptor is achemokine receptor. In the preferred embodiment of the abovecompositions, the chemokine receptor is CCR5 or CXCR4. Several otherchemokine and related receptors are known to function as HIVco-receptors including but not limited to CCR2, CCR3, CCR8, STRL33,GPR-15, CX3CR1 and APJ (69).

[0096] As used herein, “chemokine receptor” means a member of ahomologous family of seven-transmembrane spanning cell surface proteinsthat bind chemokines.

[0097] As used herein, “CCR5” is a chemokine receptor which bindsmembers of the C—C group of chemokines and whose amino acid sequencecomprises that provided in Genbank Accession Number 1705896 and relatedpolymorphic variants.

[0098] As used herein, “CXCR4” is a chemokine receptor which bindsmembers of the C—X—C group of chemokines and whose amino acid sequencecomprises that provided in Genbank Accession Number 400654 and relatedpolymorphic variants.

[0099] In one embodiment of the above compositions, at least one of thecompounds is a nonpeptidyl molecule. In one embodiment, the nonpeptidylmolecule is the bicyclam compound AMD3100. (16).

[0100] As used herein, “nonpeptidyl molecule” means a molecule that doesnot consist in its entirety of a linear sequence of amino acids linkedby peptide bonds. A nonpeptidyl molecule may, however, contain one ormore peptide bonds.

[0101] In one embodiment of the above compositions, at least one of thecompounds is an antibody. In one embodiment, the antibody is amonoclonal antibody. In another embodiment, the antibody is aanti-chemokine receptor antibody. In one embodiment, the antibody is ananti-CXCR4 antibody. In a further embodiment, the anti CXCR4 antibody is12G5. (43). In a preferred embodiment, the antibody is an anti-CCR5antibody. The anti-CCR5 antibody includes but is not limited to PA8,PA9, PA10, PA11, PA12, PA14 and 2D7. In this composition the compoundsare in an appropriate ratio. The ratio ranges from 1:1 to 1000:1.

[0102] The monoclonal antibodies PA8, PA9, PA10, PA11, PA12 and PA14were deposited pursuant to and in satisfaction of, the requirements ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209 on Dec. 2, 1998 under the following Accession Nos.: ATCCAccession No. HB-12605 (PA8), ATCC Accession No. HB-12606 (PA9), ATCCAccession No.HB-12607 (PA10), ATCC Accession No. HB-12608 (P11), ATCCAccession No. HB-12609 (PA12) ATCC Accession No. HB-12610 (PA14).

[0103] In another embodiment of the above compositions, two or more ofthe compounds are antibodies. In one embodiment of the invention, theantibodies include but are not limited to PA8, PA9, PA10, PA11, PA12,PA14 and 2D7. In this composition the antibodies are in an appropriateratio. The ratio ranges from 1:1 to 50:1.

[0104] As used herein, “antibody” means an immunoglobulin moleculecomprising two heavy chains and two light chains and which recognizes anantigen. The immunoglobulin molecule may derive from any of the commonlyknown classes, including but not limited to IgA, secretory IgA, IgG andIgM. IgG subclasses are also well known to those in the art and includebut are not limited to human IgG1, IgG2, IgG3 and IgG4. It includes, byway of example, both naturally occurring and non-naturally occurringantibodies. Specifically, “antibody” includes polyclonal and monoclonalantibodies, and monovalent and divalent fragments thereof. Furthermore,“antibody” includes chimeric antibodies, wholly synthetic antibodies,single chain antibodies, and fragments thereof. Optionally, an antibodycan be labeled with a detectable marker. Detectable markers include, forexample, radioactive or fluorescent markers. The antibody may be a humanor nonhuman antibody. The nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Methods forhumanizing antibodies are known to those skilled in the art.

[0105] As used herein, “monoclonal antibody,” also designated as mAb, isused to describe antibody molecules whose primary sequences areessentially identical and which exhibit the same antigenic specificity.Monoclonal antibodies may be produced by hybridoma, recombinant,transgenic or other techniques known to one skilled in the art.

[0106] As used herein, “anti-chemokine receptor antibody” means anantibody which recognizes and binds to an epitope on a chemokinereceptor. As used herein, “anti-CCR5 antibody” means a monoclonalantibody which recognizes and binds to an epitope on the CCR5 chemokinereceptor.

[0107] As used herein, “appropriate ratio” means mass or molar ratioswherein the compounds are synergistically effective.

[0108] In one embodiment of the above compositions, at least onecompound is a chemokine or chemokine derivative. The chemokines includebut are not limited to RANTES, MIP-1α, MIP-1β, SDF-1 or a combinationthereof. In this composition, the compounds are in an appropriate ratio.The chemokine derivatives include but are not limited to Met-RANTES,AOP-RANTES, RANTES 9-68, or a combination thereof.

[0109] As used herein, “chemokine derivative” means a chemicallymodified chemokine. The chemical modifications include but are notlimited to amino acid substitutions, additions or deletions,non-peptidyl additions or oxidations. One skilled in the art will beable to make such derivatives.

[0110] In another embodiment of the above compositions, at least onecompound is an antibody and at least one compound is a chemokine orchemokine derivative. In this composition, the compounds are in anappropriate ratio. The ratio ranges from 100:1 to 1000:1.

[0111] In another embodiment of the above compositions, at least onecompound binds to the gp41 subunit of the HIV-1 envelope glycoprotein.In one embodiment, at least one compound is the T-20 peptide inhibitorof HIV-1 entry (70).

[0112] In another embodiment of the above compositions, at least one ofthe compounds inhibits the attachment of HIV-1 to a target cell. In oneembodiment, at least one compound binds CD4. In one embodiment, at leastone compound is an HIV-1 envelope glycoprotein. In one embodiment, atleast one compound is an anti-CD4 antibody. In one embodiment, at leastone compound binds to the HIV-1 envelope glyoprotein. In one embodiment,at least one compound is an antibody to the HIV-1 envelope glycoprotein.In one embodiment, at least one compound is a CD4-based protein. In oneembodiment, at least one compound is CD4-IgG2.

[0113] In another embodiment of the above compositions, at least onecompound is an antibody and at least one compound binds to an HIV-1envelope glycoprotein. In one embodiment, the compound is a CD4-basedprotein. In one embodiment, the compound is CD4-IgG2. In thiscomposition, the compounds are in an appropriate ratio. The ratio rangesfrom 1:1 to 10:1.

[0114] As used herein, “attachment” means the process that is mediatedby the binding of the HIV-1 envelope glycoprotein to the human CD4receptor, which is not a fusion co-receptor.

[0115] As used herein, “CD4” means the mature, native, membrane-boundCD4 protein comprising a cytoplasmic domain, a hydrophobic transmembranedomain, and an extracellular domain which binds to the HIV-1 gp120envelope glycoprotein.

[0116] As used herein, “HIV-1 envelope glycoprotein” means the HIV-1encoded protein which comprises the gp120 surface protein, the gp41transmembrane protein and oligomers and precursors thereof.

[0117] As used herein, “CD4-based protein” means any protein comprisingat least one sequence of amino acid residues corresponding to thatportion of CD4 which is required for CD4 to form a complex with theHIV-1 gp120 envelope glycoprotein.

[0118] As used herein, “CD4-IgG2” means a heterotetrameric CD4-humanIgG2 fusion protein encoded by the expression vectors deposited underATCC Accession Numbers 75193 and 75194.

[0119] In one embodiment of the above compositions at least one of thecompounds comprises a polypeptide which binds to a CCR5 epitope. In oneembodiment, the epitope is located in the N-terminus, one of the threeextracellular loop regions or a combination thereof. In one embodiment,the epitope is located in the N-terminus. The epitope can comprise N13and Y15 in the N-terminus. The epitope can comprise comprises Q4 in theN-terminus. In another embodiment, the epitope includes residues in theN-terminus and second extracellular loop. The epitope can comprise D2,Y3, Q4,S7, P8 and N13 in the N-terminus and Y176 and T177 in the secondextracellular loop. The epitope can comprise D2, Y3, Q4, P8 and N13 inthe N-terminus and Y176 and T177 in the second extracellular loop. Theepitope can comprise D2 in the N-terminus and R168 and Y176 in thesecond extracellular loop. In one embodiment, the epitope is located inthe second extra cellular loop. The epitope can comprise Q170 and K171in the second extracellular loop. The epitope can comprise Q170 and E172in the second extra cellular loop.

[0120] As used herein, the following standard abbreviations are usedthroughout the specification to indicate specific amino acids: A = ala =alanine R = arg = arginine N = asn = asparagine D = asp = aspartic acidC = cys = cysteine Q = gln = glutamine E = glu = glutamic acid G = gly =glycine H = his = histidine I = ile = isoleucine L = leu = leucine K =lys = lysine M = met = methionine F = phe = phenylalanine P = pro =proline S = ser = serine T = thr = threonine W = trp = tryptophan Y =tyr = tyrosine V = val = valine

[0121] As used herein, “polypeptide” means two or more amino acidslinked by-a peptide bond.

[0122] As used herein, “epitope” means a portion of a molecule ormolecules that forms a surface for binding antibodies or othercompounds. The epitope may comprise contiguous or noncontiguous aminoacids, carbohydrate or other nonpeptidyl moities or oligomer-specificsurfaces.

[0123] As used herein, “N-terminus” means the sequence of amino acidsspanning the initiating methionine and the first transmembrane region.

[0124] As used herein, “second extra cellular loop” means the sequenceof amino acids that span the fourth and fifth transmembrane regions andare presented on the surface.

[0125] In one embodiment of the above compositions at least one of thecompounds comprises a light chain of an antibody. In another embodimentof the above compositions at least one of the compounds comprises aheavy chain of an antibody. In another embodiment of the abovecompositions at least one of the compounds comprises the Fab portion ofan antibody. In another embodiment of the above compositions at leastone of the compounds comprises the variable domain of an antibody. Inanother embodiment, the antibody is produced as a single polypeptide or“single chain” antibody which comprises the heavy and light chainvariable domains genetically linked via an intervening sequence of aminoacids. In another embodiment of the above compositions at least one ofthe compounds comprises one or more CDR portions of an antibody.

[0126] As used herein, “heavy chain” means the larger polypeptide of anantibody molecule composed of one variable domain (VH) and three or fourconstant domains (CH1, CH2, CH3, and CH4), or fragments thereof.

[0127] As used herein, “light chain” means the smaller polypeptide of anantibody molecule composed of one variable domain (VL) and one constantdomain (CL), or fragments thereof.

[0128] As used herein, “Fab” means a monovalent antigen binding fragmentof an immunoglobulin that consists of one light chain and part of aheavy chain. It can be obtained by brief papain digestion or byrecombinant methods.

[0129] As used herein, “F(ab′)2 fragment” means a bivalent antigenbinding fragment of an immunoglobulin that consists of both light chainsand part of both heavy chains. It cen be obtained by brief pepsindigestion or recombinant methods.

[0130] As used herein, “CDR” or “complementarity determining region”means a highly variable sequence of amino acids in the variable domainof an antibody.

[0131] This invention provides the above compositions and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known to those skilled in the art. Suchpharmaceutically acceptable carriers may include but are not limited toaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like.

[0132] This invention provides a method of treating a subject afflictedwith HIV-1 which comprises administering to the subject an effectivedose of the above compositions.

[0133] As used herein, “subject” means any animal or artificiallymodified animal capable of becoming HIV-infected. Artificially modifiedanimals include, but are not limited to, SCID mice with human immunesystems. The animals include but are not limited to mice, rats, dogs,guinea pigs, ferrets, rabbits, and primates. In the preferredembodiment, the subject is a human.

[0134] As used herein, “treating” means either slowing, stopping orreversing the progression of an HIV-1 disorder. In the preferredembodiment, “treating” means reversing the progression to the point ofeliminating the disorder. As used herein, “treating” also means thereduction of the number of viral infections, reduction of the number ofinfectious viral particles, reduction of the number of virally infectedcells, or the amelioration of symptoms associated with HIV-1.

[0135] As used herein, “afflicted with HIV-1” means that the subject hasat least one cell which has been infected by HIV-1.

[0136] As used herein, “administering” may be effected or performedusing any of the methods known to one skilled in the art. The methodsmay comprise intravenous, intramuscular or subcutaneous means.

[0137] The dose of the composition of the invention will vary dependingon the subject and upon the particular route of administration used.Dosages can range from 0.1 to 100,000 μg/kg. Based upon the composition,the dose can be delivered continuously, such as by continuous pump, orat periodic intervals. For example, on one or more separate occasions.Desired time intervals of multiple doses of a particular composition canbe determined without undue experimentation by one skilled in the art.

[0138] As used herein, “effective dose” means an amount in sufficientquantities to either treat the subject or prevent the subject frombecoming HIV-1 infected. A person of ordinary skill in the art canperform simple titration experiments to determine what amount isrequired to treat the subject.

[0139] This invention provides a method of preventing a subject fromcontracting HIV-1 which comprises administering to the subject aneffective dose of the above compositions.

[0140] As used herein, “contracting HIV-1” means becoming infected withHIV-1, whose genetic information replicates in and/or incorporates intothe host cells.

[0141] This invention provides an anti-CCR5 monoclonal antibody. Theantibody includes but is not limited to the following: PA8 (ATCCAccession No. HB-12605), PA9 (ATCC Accession No. HB-12606), PA10 (ATCCAccession-No. HB-12607), PA11 (ATCC Accession No. HB-12608), PA12 (ATCCAccession No. HB-12609), and PA14 (ATCC Accession No. HB-12610).

[0142] This invention provides humanized forms of the above antibodies.

[0143] As used herein, “humanized” describes antibodies wherein some,most or all of the amino acids outside the CDR regions are replaced withcorresponding amino acids derived from human immunoglobulin molecules.In one embodiment of the humanized forms of the antibodies, some, mostor all of the amino acids outside the CDR regions have been replacedwith amino acids from human immunoglobulin molecules but where some,most or all amino acids within one or more CDR regions are unchanged.Small additions, deletions, insertions, substitutions or modificationsof amino acids are permissible as long as they would not abrogate theability of the antibody to bind a given antigen. Suitable humanimmunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA andIgM molecules. A “humanized” antibody would retain a similar antigenicspecificity as the original antibody, i.e., in the present invention,the ability to bind CCR5.

[0144] One skilled in the art would know how to make the humanizedantibodies of the subject invention. Various publications, several ofwhich are hereby incorporated by reference into this application, alsodescribe how to make humanized antibodies. For example, the methodsdescribed in U.S. Pat. No. 4,816,567 (71) comprise the production ofchimeric antibodies having a variable region of one antibody and aconstant region of another antibody.

[0145] U.S. Pat. No. 5,225,539 (72) describes another approach for theproduction of a humanized antibody. This patent describes the use ofrecombinant DNA technology to produce a humanized antibody wherein theCDRs of a variable region of one immunoglobulin are replaced with theCDRs from an immunoglobulin with a different specificity such that thehumanized antibody would recognize the desired target but would not berecognized in a significant way by the human subject's immune system.Specifically, site directed mutagenesis is used to graft the CDRs ontothe framework.

[0146] Other approaches for humanizing an antibody are described in U.S.Pat. Nos. 5,585,089 (73) and 5,693,761 (74) and WO 90/07861 whichdescribe methods for producing humanized immunoglobulins. These have oneor more CDRs and possible additional amino acids from a donorimmunoglobulin and a framework region from an accepting humanimmunoglobulin. These patents describe a method to increase the affinityof an antibody for the desired antigen. Some amino acids in theframework are chosen to be the same as the amino acids at thosepositions in the donor rather than in the acceptor. Specifically, thesepatents describe the preparation of a humanized antibody that binds to areceptor by combining the CDRs of a mouse monoclonal antibody with humanimmunoglobulin framework and constant regions. Human framework regionscan be chosen to maximize homology with the mouse sequence. A computermodel can be used to identify amino acids in the framework region whichare likely to interact with the CDRs or the specific antigen and thenmouse amino acids can be used at these positions to create the humanizedantibody.

[0147] The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861(75) also propose four possible criteria which may used in designing thehumanized antibodies. The first proposal was that for an acceptor, use aframework from a particular human immunoglobulin that is unusuallyhomologous to the donor immunoglobulin to be humanized, or use aconsensus framework from many human antibodies. The second proposal wasthat if an amino acid in the framework of the human immunoglobulin isunusual and the donor amino acid at that position is typical for humansequences, then the donor amino acid rather than the acceptor may beselected. The third proposal was that in the positions immediatelyadjacent to the 3 CDRs in the humanized immunoglobulin chain, the donoramino acid rather than the acceptor amino acid may be selected. Thefourth proposal was to use the donor amino acid reside at the frameworkpositions at which the amino acid is predicted to have a side chain atomwithin 3A of the CDRs in a three dimensional model of the antibody andis predicted to be capable of interacting with the CDRs. The abovemethods are merely illustrative of some of the methods that one skilledin the art could employ to make humanized antibodies. The affinityand/or specificity of the binding of the humanized antibody may beincreased using methods of directed evolution as described in Wu et al.(1999) J. Mol. Biol. 284:151 and U.S. Pat. Nos. 6,165,793; 6,365,408 and6,413,774.

[0148] In an embodiment of the invention the humanized form of theantibody comprises a light chain variable amino acid sequence as setforth in SEQ ID NO: 6. In another embodiment, the antibody comprises aheavy chain variable amino acid sequence as set forth in SEQ ID NO: 9.In a further embodiment, the antibody may comprise the heavy chainvariable amino acid sequence as set forth in SEQ ID NO: 12.

[0149] In another embodiment, the humanized antibody comprises the lightchain variable amino acid sequence as set forth in SEQ ID NO: 6, and theheavy chain variable amino acid sequence as set forth in SEQ ID NO: 9.Alternatively, the antibody may comprise the light chain variable aminoacid sequence as set forth in SEQ ID NO: 6 and the heavy chain variableamino acid sequence as set forth in SEQ ID NO: 12.

[0150] The variable regions of the humanized antibody may be linked toat least a portion of an immunoglobulin constant region of a humanimmunoglobulin. In one embodiment, the humanized antibody contains bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3 and sometimes, CH4 region.In one embodiment, the constant regions of the humanized antibody are ofthe human IgG4 isotype.

[0151] This invention provides isolated nucleic acid molecules encodingthese anti-CCR5 monoclonal antibodies or their humanized versions. Thenucleic acid molecule can be RNA, DNA or cDNA. In one embodiment, thenucleic acid molecule encodes the light chain. In one embodiment, thenucleic acid molecule encodes the heavy chain. In one embodiment, thenucleic acid encodes both the heavy and light chains. In one embodiment,one or more nucleic acid molecules encode the Fab portion. In oneembodiment, one or more nucleic acid molecules encode CDR portions. Inone embodiment, the nucleic acid molecule encodes the variable domain.In another embodiment, the nucleic acid molecule encodes the variabledomain and one or more constant domains.

[0152] Preferably, analogs of exemplified humanized anti-CCR5 antibodiesdiffer from exemplified humanized anti-CCR5 antibodies by conservativeamino acid substitutions. For purposes of classifying amino acidsubstitutions as conservative or non-conservative, amino acids may begrouped as follows: Group I (hydrophobic side chains): met, ala, val,leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr;Group III (acidic side chains): asp, glu; Group IV (basic side chains):asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

[0153] Analogs of humanized anti-CCR5 antibodies show substantial aminoacid sequence identity with humanized PRO 140 #1 or humanized PRO 140#2, exemplified herein. Heavy and light chain variable regions ofanalogs are encoded by nucleic acid sequences that hybridize with thenucleic acids encoding the heavy or light chain variable regions ofhumanized PRO 140 #1, or humanized PRO 140 #2, or degenerate formsthereof, under stringent conditions.

[0154] Due to the degeneracy of the genetic code, a variety of nucleicacid sequences encode the humanized anti-CCR5 antibody of the presentinvention. In certain embodiments, the antibody is encoded by a nucleicacid molecule that is highly homologous to the foregoing nucleic acidmolecules. Preferably the homologous nucleic acid molecule comprises anucleotide sequence that is at least about 90% identical to thenucleotide sequence provided herein. More preferably, the nucleotidesequence is at least about 95% identical, at least about 97% identical,at least about 98% identical, or at least about 99% identical to thenucleotide sequence provided herein. The homology can be calculatedusing various, publicly available software tools well known to one ofordinary skill in the art. Exemplary tools include the BLAST systemavailable from the website of the National Center for BiotechnologyInformation (NCBI) at the National Institutes of Health.

[0155] One method of identifying highly homologous nucleotide sequencesis via nucleic acid hybridization. Thus the invention also includeshumanized CCR5 antibodies having the CCR5-binding properties and otherfunctional properties described herein, which are encoded by nucleicacid molecules that hybridize under high stringency conditions to theforegoing nucleic acid molecules. Identification of related sequencescan also be achieved using polymerase chain reaction (PCR) and otheramplification techniques suitable for cloning related nucleic acidsequences. Preferably, PCR primers are selected to amplify portions of anucleic acid sequence of interest, such as a CDR.

[0156] The term “high stringency conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989, or Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. One example ofhigh stringency conditions is hybridization at 65 degrees Centigrade inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄ (pII7), 0.5%SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate,pH7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, a membrane uponwhich the nucleic acid is transferred is washed, for example, in 2×SSCat room temperature and then at 0.1-0.5×SSC/0.1×SDS at temperatures upto 68 degrees Centigrade.

[0157] The nucleic acid sequences are expressed in hosts after thesequences have been operably linked to (i.e., positioned to ensure thefunctioning of) an expression control sequence. These expression vectorsare typically replicable in the host organisms, either as episomes or asan integral part of the host chromosomal DNA. Commonly, expressionvectors will contain selection markers, e.g., tetracycline or neomycin,to permit detection of those cells transformed with the desired DNAsequences (see, e.g., U.S. Pat. No. 4,704,362 which is incorporatedherein by reference).

[0158]E. coli is one prokaryotic host useful particularly for cloningthe DNA sequences of the present invention. Other microbial hostssuitable for use include bacilli, such as Bacillus subtilus, and otherenterobacteriaccae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

[0159] Other microbes, such as yeast, may also be useful for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes and an origin ofreplication, termination sequences and the like as desired.

[0160] In addition to microorganisms, mammalian tissue cell culture mayalso be used to express and produce the polypeptides of the presentinvention (see, Winnacker, “From Genes to Clones,”, VCH Publishers, NewYork, N.Y. (1987)). Eukaryotic cells are actually preferred, because anumber of suitable host cell lines capable of secreting intactimmunoglobulins have been developed in the art, and include the CHO celllines, various COS cell lines, HeLa cells, preferably myeloma celllines, etc. and transformed B cells or hybridomas. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen, et al., Immunol.Rev., 89, 49-68 (1986) which is incorporated herein by reference), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom immunoglobulin genes, SV40, Adenovirus, cytomegalovirus, BovinePapilloma Virus, and the like.

[0161] The vectors containing the DNA segments of interest (e.g., theheavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment or electroporation may be used forother cellular hosts (see generally, Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press (1982) which isincorporated herein by reference).

[0162] Once expressed, the whole antibodies, their dimers, individuallight and heavy chains, or other immunoglobulin forms of the presentinvention, can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like (see generally, R.Scopes, “Protein Purification”, Springer-Verlag, New York (1982)).Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(see generally, Immunological Methods, Vols. I and II, Lefkovits andPernis, eds., Academic Press, New York, N.Y. (1979 and 1981)).

[0163] For diagnostic or detection purposes, the antibodies may eitherbe labeled or unlabeled. Unlabeled antibodies can be used in combinationwith other labeled antibodies (second antibodies) that are reactive withthe humanized antibody, such as antibodies specific for humanimmunoglobulin constant regions. Alternatively, the antibodies can bedirectly labeled. A wide variety of labels can be employed, such asradionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands (particularly haptens), etc. Numerous typesof immunoassays are available and are well known to those skilled in theart for detection of CCR5-expressing cells or detection of CCR5modulation on cells capable of expressing CCR5.

[0164] The present invention also provides antibody fragment-polymerconjugates having an effective size or molecular weight that confers anincrease in serum half-life, an increase in mean residence time incirculation (MRT) and/or a decrease in serum clearance rate overunderivatized antibody fragments.

[0165] The antibody fragment-polymer conjugates of the invention can bemade by derivatizing the desired antibody fragment with an inertpolymer. It will be appreciated that any inert polymer which providesthe conjugate with the desired apparent size or which has the selectedactual molecular weight is suitable for use in constructing the antibodyfragment-polymer conjugates of the invention.

[0166] Many inert polymers are suitable for use in pharmaceuticals. See,e.g., Davis et al., Biomedical Polymers: Polymeric Materials andPharmaceuticals for Biomedical Use, pp. 441-451 (1980). In allembodiments of the invention, a non-protinaceous polymer is used. Thenonprotinaceous polymer ordinarily is a hydrophilic synthetic polymer,i.e., a polymer not otherwise found in nature. However, polymers whichexist in nature and are produced by recombinant or in vitro methods arealso useful, as are polymers which are isolated from native sources.Hydrophilic polyvinyl polymers fall within the scope of this invention,e.g., polyvinylalcohol and polyvinvypyrrolidone. Particularly useful arepolyalkylene ethers such as polyethylene glycol (PEG); polyoxyalklyenessuch as polyoxyethylene, polyoxypropylene and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides which comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g., polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g., hyaluronic acid, polymers of sugar alcoholssuch as polysorbitol and polymannitol, heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble butthe final conjugate must be water soluble. Preferably, the conjugateexhibits a water solubility of at least about 0.01 mg/ml and morepreferably at least about 0.1 mg/ml, and still more preferably at leastabout 1 mg/ml. In addition the polymer should not be highly immunogenicin the conjugate form, nor should it possess viscosity that isincompatible with intraveneous infusion or injection if the conjugate isintended to be administered by such routes.

[0167] In one embodiment, the polymer contains only a single group whichis reactive. This helps to avoid cross-linking of protein molecules.However it is within the scope of the invention to maximize reactionconditions to reduce cross-linking, or to purify the reaction productsthrough gel filtration or ion-exchange chromatography to recoversubstantially homogeneous derivatives. In other embodiments the polymercontains two or more reactive groups for the purpose of linking multipleantibody fragments to the polymer backbone.

[0168] Again, gel filtration or ion-exchange chromatography can be usedto recover the desired derivative in substantially homogeneous form.

[0169] The molecular weight of the polymer can range up to about 500,000D and preferably is at least about 20,000 D, or at least about 30,000 D,or at least about 40,000 D. The molecular weight chosen can depend uponthe effective size of the conjugate to be achieved, the nature (e.g.,structure such as linear or branched) of the polymer and the degree ofderivitization, i.e., the number of polymer molecules per antibodyfragment, and the polymer attachment site or sites on the antibodyfragment.

[0170] The polymer can be covalently linked to the antibody fragmentthrough a multifunctional crosslinking agent which reacts with thepolymer and one or more amino acid residues of the antibody fragment tobe linked. However, it is also within the scope of the invention todirectly crosslink the polymer by reacting a derivatized polymer withthe antibody fragment, or vice versa.

[0171] The covalent crosslinking site on the antibody fragment includesthe N-terminal amino group and epsilon amino groups found on lysineresidues, as well other amino, imino, carboxyl, sulfhydryl, hydroxyl orother hydrophilic groups. The polymer may be covalently bonded directlyto the antibody fragment without the use of a multifunctional(ordinarily bifunctional) crosslinking agent, as described in U.S. Pat.No. 6,458,355.

[0172] The degree of substitution with such a polymer will varydepending upon the number of reactive sites on the antibody fragment,the molecular weight, hydrophilicity and other characteristics of thepolymer, and the particular antibody fragment derivitization siteschosen. In general, the conjugate contains from 1 to about 10 polymermolecules, but greater numbers of polymer molecules attached to theantibody fragments of the invention are also contemplated. The desiredamount of derivitization is easily achieved by using an experimentalmatrix in which the time, temperature and other reaction conditions arevaried to change the degree of substitution, after which the level ofpolymer substitution of the conjugates is determined by size exclusionchromatography or other means known in the art.

[0173] Functionalized PEG polymers to modify the antibody fragments ofthe invention are available from Shearwater Polymers, Inc. (Huntsville,Ala.). Such commercially available PEG derivatives include, but are notlimited to, amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol,PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG aminoacids, PEG succinimidyl succinate, PEG succinimidyl propionate,succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate ofPEG, succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole,PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether,PEG-aldehyde, PEG-vinylsulfone, PEG-maleimide,PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinylderivatives, PEG silanes and PEG phospholides. The reaction conditionsfor coupling these PEG derivatives will vary depending on the protein,the desired degree of PEGylation and the PEG derivative utilized. Somefactors involved in the choice of PEG derivatives include: the desiredpoint of attachment (such as lysine or cysteine R-groups), hydrolyticstability and reactivity of the derivatives, stability, toxicity andantigenicity of the linkage, suitability for analysis, etc. Specificinstructions for the use of any particular derivative are available fromthe manufacturer. The conjugates of this invention are separated fromthe unreacted starting materials by gel filtration or ion exchange HPLC.

[0174] The anti-CCR5 antibody or fragments thereof may be used incombination with one or more additional anti-viral agents selected fromthe group consisting of nonnucleoside reverse transcriptase inhibitors(NNRTIs), a nucleoside reverse transcriptase inhibitor, an HIV-1protease inhibitor, a viral entry inhibitor and combinations thereof.

[0175] The known NNRTI compounds that may be used in the composition ofthe present invention include but are not limited to efavirenz, UC-781,HBY 097, nevirapine(11-cyclopropyl-5,11,-dihydro-4-methyl-6H-dipyrido[3,2-b:2′3′-][1,4]diazepin-6-one),delavirdine ((Rescriptor™; Pharmacia Upjohn) (piperazine,1-[3-[(1-methyl-ethyl)amino]-2-pyridinyl]-4-[[5-[(methysulfonyl)amino]-1H-indol-2-yl]carbonyl]-,monomethanesulfonate),SJ-3366(1-(3-cyclopenten-1-yl)methyl-6-(3,5-dimethylbenzoyl)-5-ethyl-2,4-pyrimidinedione),MKC-442 (6-benzyl-1-(ethoxymethyl)-5-isopropyluracil), GW420867x (S-3ethyl-6-fluro-4-isopropoxycarbonyl-3,4-dihydro-quinoxalin-2(1H)-one;Glaxo),HI-443(N′-[2-(2-thiophene)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea), andthe like.

[0176] The nucleoside reverse transcriptase inhibitors that may be usedin the composition in combination with at least one anti-CCR5 antibodyor fragment thereof of the present invention include but are not limitedto abacavir (Ziagen™, GlaxoSmithKline)((1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsulfate (salt)), lamivudine (Epivir™, GlaxoSmthKline) ((2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one),zidovudine (Retrovir™; GlaxoSmithKline) (3′azido-3′-deoxythymidine),stavudine (Zerit; Bristol-Myers Squibb)(2′,3′-didehydro-3′deoxythymidine), zacitabine (Hivid™; RocheLaboratories)(4-amino-1-beta-D2′,3′-dideoxyribofuranosyl-2-(1H)-pyrimidone),didanosine, and the like.

[0177] The HIV-1 protease inhibitors that may be used in the compositionin combination with anti-CCR5 antibody or fragments thereof of thepresent invention include but are not limited to lopinavir (1S-[1R*,(R*),3R*,4R*]]-N-4-[[(2,6-dimethyphenoxy)acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha-(1-methylethyl)-2-oxo1(2H)-pyrimidineacetamide),saquinavir(N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-(3S)-carboxamide),nelfinavir mesylate([3S-[2(2S*,3S*),3a,4β,8aβ]]-N-(1,1-dimethyetyl)decahydro-2[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamidemono-methane sulfonate), indinavir sulfate(([1(1S,2R),5(S))]-2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(1,1-dimethylethyl)amino]carbonyl]-4-(3-pyridinylmethyl)-1-piperazinyl]-2-(phenylmethyl)-D-erythropentonamidesulfate (1:1) salt), amprenavir ((3S)-tetrahydro-3-furylN-[(1S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl-2-hydroxypropyl]carbamate),ritonavir((10-Hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid,5-thiazolylmethylester, [5S-(5R*, 8R*, 10R*, 11R*)]), and the like.

[0178]

[0179] HIV-1 fusion or viral entry inhibitors that may be used incombination with the anti-CCR5 antibody or fragments thereof of thepresent invention include PRO 542 (Progenics Pharmaceuticals, Inc.,Tarrytown, N.Y.), T-20 (Trimeris, Inc., Durham, N.C.) (U.S. Pat. Nos.5,464,933; 6,133,418; 6,020,459), T-1249 (U.S. Pat. Nos. 6,345,568;6,258,782), and the like.

[0180] For combination therapy, the anti-CCR5 antibody or fragmentthereof of the present invention may be provided to the subject priorto, subsequent to, or concurrently with one or more conventionalantiviral agents.

[0181] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0182] Experimental Details:

EXAMPLE 1

[0183] A. Materials and Methods

[0184] 1) Reagents

[0185] MAb 2D7 was purchased from Pharmingen (San Diego, Calif.) and CC-and CXC-chemokines were obtained from R&D Systems (Minneapolis, Minn.).CD4-IgG2 (1), soluble (s) CD4 (2) and recombinant HIV-1_(JR-FL) gp120,were produced by Progenics Pharmaceuticals, Inc. (59).

[0186] 2) Isolation and Purification of Anti-CCR5 mAbs

[0187] L1.2-CCR5⁺ cells (63) were incubated for 16 h in the presence of5 mM sodium butyrate, which activates transcription from thecytomegalovirus (CMV) promoter that controls CCR5 expression, resultingin a 10-fold increase in cell surface co-receptor density. Female Balb/cmice were immunized intraperitoneally with 10⁷ L1.2-CCR5⁺ cells at3-week intervals, and administered an intravenous boost of 10⁷L1.2-CCR5⁺ cells three days prior to splenectomy. Splenocytes were fusedwith the Sp2/0 cell line. In a primary screen, supernatants from tenthousand hybridoma cultures were tested; one hundred and twenty of theseinhibited HIV-1 envelope-mediated fusion between PM1 cells (10), whichnaturally express CCR5 and CD4, and HeLa-Env_(JR-FL) ⁺ cells in aresonance energy transfer (RET) assay, as previously described (19, 38).Hybridomas that produced the most potently inhibitory supernatants andthat also stained CCR5⁺ cells were sub-cloned by limiting dilution.Ascites fluids were prepared by Harlan Bioproducts for Science, Inc.(Indianapolis, Ind.) from Balb/c mice that were injected with hybridomasproducing the anti-CCR5 mAbs PAB, PA9, PA10, PA11, PA12 and PA14. ThemAbs were individually purified to >95% homogeneity by precipitationwith ammonium sulfate followed by protein-A chromatography. All mAbswere resuspended in phosphate buffered saline (PBS) at a finalconcentration of 5 mg/ml.

[0188] 3) Fluorescence Activated Cell Sorting (FACS) Analysis andEpitope Mapping of Anti-CCR5 mAbs

[0189] Flow cytometry was used to detect cell-surface reactivity of mAbsPA8-PA12 and PA14 with CCR5. Sodium butyrate treated L1.2-CCR5⁺ cells(10⁶) were incubated with 0.25 μg of antibody, for 20 min at 4° C. in0.1% sodium azide (NaN₃) in 50 μl of Dulbecco's PBS (DPBS). The CCR5 mAb2D7 was used as a positive control, a non-specific murine IgG1 was usedas a negative control. The cells were spun down, washed and incubatedwith phycoerythrin (PE)-labeled goat anti-mouse IgG (Caltag, Burlingame,Calif.) diluted 1:100, under the same conditions as the first antibodyincubation. Finally, cells were analyzed by flow cytometry. PBMC wereisolated and stimulated as previously described (60) and stained usingsimilar methods.

[0190] A similar procedure was used for epitope mapping of the anti-CCR5mAbs. A panel of seventy CCR5 point mutants has been described (20, 24,52). The coding sequences of these proteins are sub-cloned into thepcDNA3.1 vector (Stratagene) from which transcription can be driven by a5′ T7-polymerase promoter. The CCR5 mutants carry a 9-residuehemaglutinin (HA) tag at the C-terminus for detection of protein in celllysates or by flow cytometry. HeLa cells (2×10⁶) were incubated for 5 hwith 20 μg/ml lipofectin and an equal amount of wild-type or mutantCCR5-expressing plasmid in OPTI-MEM (Life Technologies, Gaithersburg,Md.). The cells were then infected for 12 h with 2×10⁷ p.f.u. of vTF7(23) to boost CCR5 expression, detached with 2 mM ethylenediaminetetracetic acid (EDTA) in PBS and washed once with binding buffer (1%BSA, 0.05% NaN₃ in DPBS). Cells (1×10⁶) were surface labeled with mAbsas described in the previous paragraph, washed once with the incubationbuffer and resuspended in 1 ml of 1×FACSlyse in water (Becton Dickinson)for 30 min at room temperature, to permeabilize the cell membranes. Thecells were then spun down, washed with the incubation buffer andincubated for 1 h at 37° C. with 4 μg/ml of a fluorescein isothiocyanate(FITC)-labeled mouse anti-HA mAb (BabCo, Richmond, Calif.) forintracellular labeling. Finally, cells were washed once with bindingbuffer and once with DPBS, resuspended in 1% formaldehyde in PBS andanalyzed by flow cytometry. The extent of binding of a mAb to mutantCCR5 was determined by the equation (mutant CCR5 PE m.f.i./wt CCR5 PEm.f.i.)/(mutant CCR5 FITC m.f.i./wt CCR5 FITC m.f.i.)×100%. Thisnormalizes mAb binding for mutant co-receptor expression levels.

[0191] 4) gp120/sCD4-Binding Assay

[0192] gp120 was bibtinylated using NHS-biotin (Pierce, Rockford, Ill.)according to the manufacturer's instructions, and uncoupled biotin wasremoved by diafiltration. Sodium butyrate-treated L1.2-CCR5⁺ cells wereincubated with varying dilutions of an equimolar mixture of sCD4 andbiotinylated gp120, or 1.25 μg/ml of sCD4 and 2.5 μg/ml of biotinylatedgp120 in the presence of varying concentrations of anti-CCR5 mAbsPA8-PA12, PA14, 2D7 or a non-specific murine IgG1, for 1 h at roomtemperature in 0.1% NaN₃in DPBS. Cells were washed with the incubationbuffer and incubated with streptavidin-PE (Becton Dickinson) diluted1:50, for 1 h at room temperature. Finally, cells were washed withbinding buffer and analyzed using a fluorescence plate reader(Perspective Biosystems, Framingham, Mass.).

[0193] 5) Inhibition of Envelope-Mediated Cell-Cell Fusion and HIV-1Entry by Anti-CCR5 mAbs

[0194] HIV-1 envelope-mediated fusion between HeLa-Env_(JR-FL) ⁺ and PM1cells was detected using the RET assay. Equal numbers (2×10⁴) offluorescein octadecyl ester (F18)-labeled envelope-expressing cells andoctadecyl rhodamine (R18)-labeled PM1 cells were plated in 96-wellplates in 15% fetal calf serum in DPBS and incubated for 4 h at 37° C.in the presence of varying concentrations of the anti-CCR5 mAbs,PA8-PA12, PA14, 2D7 or a non-specific murine IgG1. Fluorescence RET wasmeasured with a Cytofluor plate-reader (PerSeptive Biosystems) and % RETwas determined as previously described (38).

[0195] NLluc⁺env⁻ viruses complemented in trans by envelopeglycoproteins from JR-FL or Gun-1 were produced as previously described(20). U87MG-CD4⁺CCR5⁺ cells (14) were infected with chimeric, reporterviruses containing 50-100 ng/ml p24 in the presence of varyingconcentrations of the individual mAbs. After 2 h at 37° C.,virus-containing media were replaced by fresh, mAb-containing media.Fresh media, without antibodies, were added again after 12 hours. Aftera total of 72 h, 100 μl of lysis buffer (Promega) were added to thecells and luciferase activity (r.l.u.) was measured as described (20).The % inhibition of HIV-1 infection is defined as [1−(r.l.u in thepresence of antibody/r.l.u in the absence of antibody)]×100%.

[0196] 6) Calcium Signaling Assays

[0197] The fluorochrome Indo-1AM (Molecular Probes, Eugene, Oreg.) wasadded to sodium butyrate treated L1.2-CCR5⁺ cells at a finalconcentration of 5 μM. After incubation at 37° C. for 30 min, the cellswere washed once and resuspended in Hank's buffered saline. Cells (10⁶)were stimulated sequentially with an anti-CCR5 mAb or PBS, followed 60slater with RANTES. MAbs PA8-PA12 and PA14 were used at a concentrationof 100 μg/ml, 2D7 at 20 μg/ml and RANTES at 250 ng/ml. Calcium fluxinhibition by PA14 and 2D7 was also tested for a wide range of mAbconcentrations, ranging from 0-100 μg/ml. Intracellular calcium levelswere monitored using a Perkin-Elmer LS-50S fluorescencespectrophotometer by measuring the ratio of fluorescence emissions at402 nm (bound dye) and 486 nm (free dye) following excitation at 358 nm.

[0198] B. Results and Discussion

[0199] 1) Isolating Anti-CCR5 Monoclonal Antibodies PA8, PA9, PA10,PA11, PA12 and PA14

[0200] It was found that peptides corresponding to the extracellulardomains of CCR5 are inefficient at raising specific, high-titer antibodyresponses against the native, cell surface receptor (50). Balb/C micewere immunized, therefore, with L1.2-CCR5⁺ cells and hybridoma culturesupernatants were tested for their ability to inhibit JR-FLenvelope-mediated membrane fusion with CD4⁺CCR5⁺ PM1 cells in the RETassay (19, 38). Even though well over a hundred supernatants inhibitedcell-cell fusion by >50%, only six—designated PAB, PA9, PA10, PA11, PA12and PA14—specifically and intensely stained L1.2-CCR5⁺ but not theparental L1.2 cells, as demonstrated by flow cytometry (data not shown).Based on previous experience, it was assumed that the other mAbs capableof inhibiting cell-cell fusion were probably directed against cellsurface adhesion molecules such as LFA-1 (37). Hybridomas PA8-PA12 andPA14 were determined by isotyping ELISA (Cappell, Durham, N.C.) tosecrete IgG1 mAbs. Ascites fluids were prepared from Balb/C mice thatwere injected with the six hybridomas and the IgG1 fractions werepurified. PA6, PA9, PA11, PA12 and PA14 exhibited distinct isoelectricfocussing profiles, whereas PA10 had a very similar profile to that ofPA9 and therefore may be a second isolate of the same mAb (data notshown).

[0201] 2) MAb Binding to CCR5+ Cells

[0202] None of the purified anti-CCR5 mAbs stained the parental L1.2cell line (data not shown). However, mAbs PA9-PA12 and PA14stained >90%, and PA8 stained ˜70%, of L1.2-CCR5⁺ cells as determined byflow cytometry, showing they recognized CCR5 (FIG. 1). The anti-CCR5 mAb2D7, which was a positive control in our experiments, also stained >90%of L1.2-CCR5⁺ cells. PA8-PA12 and PA14 are all IgG1, and react equallywell with a goat anti-mouse IgG, whereas 2D7 is an IgG2a and may reactdifferently with the reporter antibody. Only mean fluorescenceintensities (m.f.i.) measured with mAbs PA8-PA12 and PA14 therefore aredirectly comparable. The rank order of mean fluorescence intensities(m.f.i.) was PA12˜PA11>(2D7=) PA14˜PA10˜PA9>PA8. The difference betweenPA12 m.f.i. and PA8 m.f.i. was three-fold. Differences in stainingintensity between PA8 and the other mAbs remained constant over a widerange of concentrations (data not shown) and probably do not correspondto differences in mAb affinities for CCR5. This implies that PA8interacts only with a subset of CCR5 molecules present on the surface ofL1.2-CCR5⁺ cells.

[0203] Compared with L1.2-CCR5+ cells, mitogen-stimulated PBMC exhibiteddifferent patterns of staining by the anti-CCR5 mAbs. 2D7 and PA14stained>20%, PA11 and PA12 stained ˜10%, PA8, PA9 and PA10 stained<5% ofPBMC (FIG. 1). The mean fluorescence intensities of the stained PBMCwere about ten-fold lower than those obtained with L1.2-CCR5⁺ cells foreach mAb; their rank order was (2D7>) PA14>PA12˜PA11˜PA10˜PA9˜PA8.Again, this differed somewhat from the order of reactivities observed onCCR5 transfectants. The difference between PA9 m.f.i. and PA14 m.f.i.was seven-fold. Other groups have observed similar differences in theability of anti-CCR5 mAbs to stain stable, CCR5⁺ cell lines versus PBMC(28). This may be due to cell-specific differences in CCR5 conformation,post-translational modification or oligomerization. Alternatively,association with other cell surface molecules may differ between cells.Since an obvious choice for such a molecule would be the CD4 cellsurface antigen, which is absent from L1.2-CCR5⁺ cells and present onPBMCs, we also tested the ability PA8-PA12, PA14 and 2D7 to stain HeLacells transiently expressing CCR5 alone-or with CD4. No differences wereobserved in the ability of any of the mAbs to stain cell surface CCR5 inthe presence of CD4 (data not shown). If there is an association betweenthese two proteins, it does not involve epitopes recognized by theanti-CCR5 mAbs available to us. Alternatively, an association betweenCCR5 and CD4 might only occur on primary lymphocytes.

[0204] 3) Epitope Mapping of the mAbs Using CCR5 Alanine Mutants

[0205] None of the antibodies were able to detect reduced and denaturedCCR5 protein by Western blotting indicating that they recognizeconformationally sensitive epitopes (data not shown). MAb epitopemapping studies were performed using a panel of seventy alanine pointmutants of residues in the Nt and ECLs of CCR5. HeLa cells werelipofected with mutant or wild type CCR5 coding sequences appended withC-terminal HA tags, and infected with vTF7 (23) to boost co-receptorexpression. The cells were then incubated with the anti-CCR5 mAbs andtheir binding was revealed by a PE-labeled goat anti-mouse IgG. Asecond, intracellular stain was performed with a FITC-labeled anti-HAmAb (BabCo). This internal control allowed us to directly normalizestaining by the anti-CCR5 MAbs for mutant co-receptor expression levelson the cell surface. Hence, mAb binding to each mutant is expressed as apercentage of binding to wild-type CCR5 (FIG. 4).

[0206] Certain point mutations reduced the binding of all of theantibodies to CCR5 by >50%. In general, PA8-PA12 were the most affected,PA14 and 2D7 the least affected by this class of mutants, which includedthe cysteine pair C101A and C178A, the Nt mutants Y10A, D11A, K25A, theECL1 mutant D95A, the ECL2 mutants K171A/E172A, Q188A, K191A/N192A, andthe ECL3 mutants F263A and F264A (FIG. 1). One interpretation is thatthese residues are not part of the mAb epitopes per se, but thatchanging them to alanines causes conformational perturbations that havea common effect on binding of all mAbs. We assumed that if a mutationlowered binding of an individual mAb by >75%, and did not also lowerbinding of most of the other antibodies, the residue was probably adirect contributor to the epitope recognized by the mAb. Using thesestringent guidelines, it was concluded that the seven anti-CCR5 mAbsrecognize overlapping but distinct epitopes (FIG. 4). MAb PA8 binding toCCR5 depended on N13 and Y15 in the Nt. MAb PA9 and PA10 required D2,Y3, Q4, P8 and N13 in the Nt, and Y176 and T177 in ECL2. MAb PA9 alsorequired S7 in the Nt. MAb PA11 and PA12 binding depended on Q4 in theNt. PA14 required D2 in the Nt, and R168 and Y176 in ECL2. Finally, mAb2D7 required Q170 and K171/E172 in ECL2 in order to bind to CCR5.

[0207] 4) Chemokine Signaling in the Presence of anti-CCR5 mAbs

[0208] Chemokine receptor-binding agents can be antagonists or, morerarely, agonists of receptor-mediated intracellular signaling.Alternatively, they could have no effect on signaling. CCR5 is able tobind three CC-chemokines, RANTES, MIP-1α and MIP-1β and transduce asignal that modulates cytosolic calcium levels. We therefore tested theagonist/antagonist activity of various concentrations of mAbs PA8-PA12,PA14 and 2D7. Changes in intracellular calcium concentrations, (Ca²⁺)i,were measured in Indo-1-loaded L1.2-CCR5⁺ cells. None of the mAbsstimulated a change in (Ca²⁺)i, indicating that they are not agonistsfor CCR5. PA8-PA12 were also unable to inhibit Ca²⁺ fluxes induced byRANTES (FIG. 5A and data not shown), even at concentrations as high as100 μg/ml, showing they are not antagonists either. These concentrationsprovide saturating binding of the mAbs to L1.2-CCR5⁺ cells, as shown byflow cytometry and the gp120/CCR5 binding assay (FIG. 6D and data notshown). MAbs PA14 and 2D7, however, blocked calcium mobilization inducedby RANTES, although with different potencies (FIGS. 5A, 5B). The IC₅₀for PA14 calcium influx inhibition was 50 μg/ml, which was approximately8-fold higher than the IC₅₀ for 2D7 (FIG. 5B). RANTES-, MIP-1α- andMIP-1β-induced calcium fluxes were each inhibited by similarconcentrations of PA14 (data not shown). None of the mAbs affectedSDF-1-induced calcium mobilization in L1.2-CCR5⁺ cells, whichendogenously express CXCR4 (data not shown). Finally, neither mAbs norCC-chemokines affected cytosolic calcium levels in parental L1.2 cells(data not shown).

[0209] 5) Inhibition of CCR5 Co-Receptor Function by the mAbs

[0210] MAbs PA8-PA12 and PA14 were initially selected on the basis oftheir ability to inhibit HIV-1 envelope-mediated cell-cell fusion. Thisactivity was confirmed and quantified for the purified mAbs. Asexpected, all six mAbs, as well as mAb 2D7, blocked fusion betweenCD4⁺CCR5⁺ PM1 cells and HeLa-Env_(JR-FL) ⁺ cells in the RET assay. Therank order of potency was 2D7˜PA14>PA12>PA11>PA10˜PA9˜PA8 (FIG. 6A).IC₅₀ values for PA14 and 2D7 were 1.7 μg/ml and 1.6 μg/ml respectively,for PA11 and PA12 these were 25.5 μg/ml and 10.0 μg/ml respectively(FIG. 3). PA8, PA9 and PA10 inhibited fusion by only 10-15% at 300μg/ml. None of the mAbs affected fusion between PM1 cells andHeLa-Env_(LAI) ⁺ cells, which express the full length envelope proteinfrom an X4 virus (data not shown).

[0211] The ability of the different anti-CCR5 mAbs to inhibit entry of aprototypic R5 virus, JR-FL, and a R5X4 virus, Gun-1, in a single-roundof replication, luciferase-based entry assay was also tested. The rankorder of potency in the entry assay was similar to the one determined inthe cell-cell fusion assay (FIG. 6B) A >50% inhibition of JR-FL or Gun-1entry with PA8-PA11 was unable to be obtained. The IC₅₀ value for PA12was 2.5 μg/ml. However, inhibition of entry by >60% with this mAb wasunable to be obtained. The IC₅₀ values for PA14 and 2D7 inhibition ofJR-FL entry were determined to be 0.024 and 0.026 μg/ml respectively(FIG. 3), and were 60-fold lower then those obtained in the fusionassay. Entry of dual-tropic Gun-1 was 2-3-fold more sensitive toinhibition by anti-CCR5 mAbs than JR-FL entry (data not shown).

[0212] Anti-co-receptor mAbs might inhibit envelope-mediated fusioneither by directly affecting the gp120/CCR5 interaction or by impedingpost-binding steps involved in the formation of an active fusioncomplex. To determine the mechanism of inhibition of viral fusion andentry by PA8-PA12 and PA14, the ability of the different mAbs to blockthe gp120/CCR5 interaction was tested. For this an assay that detectsbinding to L1.2-CCR5⁺ cells of biotinylated HIV-1_(JR-FL) gp120complexed with sCD4 was used. No binding of biotinylated gp120 wasobserved in the absence of sCD4 or CCR5, or when HIV-1_(LAI) gp120 wasused (FIG. 6C).

[0213] With the exception of PA8, all mAbs abrogated gp120/sCD4 bindingto L1.2-CCR5⁺ (FIG. 6D) . Inhibition by PA8 saturated at ˜40%, whichconcurs with flow cytometry data (FIG. 1) in suggesting that this mAbbinds only to a subset of CCR5 molecules on L1.2-CCR5⁺ cells. MAbs PA9,PA10, PA11 and PA12 inhibited binding with IC₅₀ values of 0.24, 0.13,0.33, 0.24 μg/ml respectively (FIG. 3). Surprisingly, mAbs PA14 and 2D7were the two least efficient inhibitors of gp120/sCD4 binding, with IC₅₀values of 1.58 and 1.38 μg/ml respectively (FIG. 3). Therefore, therewas no correlation between the ability of a mAb to inhibitgp120/CD4/CCR5-mediated membrane fusion and entry and its ability toblock gp120/sCD4 binding to the co-receptor.

[0214] 6) Synergistic Inhibition of HIV-1 Fusion by Combinations ofAnti-CCR5 mAbs and Other Viral Entry Inhibitors

[0215] Co-receptor-specific agents may act at multiple stages of theentry process and exhibit non-additive effects when used in combination.From a clinical perspective, it is important to determine theinteractions of co-receptor-specific drug candidates with endogenouschemokines, which may afford some level of protection against diseaseprogression. CCR5 mAbs were therefore tested in combination with eachother or with RANTES, or with CD4-IgG2, which binds to HIV-1 gp120 toinhibit attachment to target cells. Dose-response curves were obtainedfor the agents used individually and in combination in viral fusion andentry assays. Data were analyzed using the median effect principle (9).The concentrations of single-agents or their mixtures required toproduce a given effect were quantitatively compared in a term known asthe Combination Index (CI). A CI value greater than 1 indicatesantagonism, CI˜1 indicates an additive effect, and CI<1 indicates asynergistic effect wherein the presence of one agent enhances the effectof another.

[0216] Combinations of PA12 and 2D7 were the most potently synergistic,with CI values ranging between 0.02 and 0.29, depending on the ratio ofthe antibodies (FIG. 7 and FIG. 2). The degree of synergy is known tovary with the stoichiometry of the agents. The viral entry and fusionassays were generally consistent in identifying mAb combinations thatare highly synergistic, PA12 and 2D7; moderately synergistic, PA12 andPA14; additive, PA11 and PA12; and weakly antagonistic, PA14 and 2D7.The lack of synergy between PA14 and 2D7 is not surprising given thatthese mAbs cross-compete for binding to CCR5⁺ cells as determined byflow cytometry (data not shown). The observation of an additive effectof PA11 and PA12 may be an indication that these mAbs bind to slightlydifferent epitopes in CCR5, while sharing a dependency on residue Q4 inthe Nt.

[0217] The ability of mAbs PA12, PA14 and 2D7 to synergize with RANTESin blocking cell-cell fusion was also tested. PA12 and RANTEScombinations exhibited moderate synergy (FIG. 2). PA14 and 2D7 exhibitedno synergy with RANTES, which is consistent with these mAbs beinginhibitory of RANTES binding and signaling (FIGS. 5A, 5B). Finally, wetested synergy between mAbs PA12, PA14, 2D7 and CD4-IgG2, whichinteracts with gp120 . We observed moderate synergy between PA12 andCD4-IgG2 but no synergy between PA14 or 2D7 and CD4-IgG2 (FIG. 2).

[0218] Experimental Discussion

[0219] Six murine anti-CCR5 IgG1 mAbs were isolated and characterized.Whereas PA8, PA9, PA11, PA12 and PA14 are distinct molecular species,PA9 and PA10 are indistinguishable by the analyses and therefore areprobably the same mAb. All of the mAbs that were isolated recognizecomplex conformational epitopes, as is often the case with mAbs raisedagainst native, cell surface proteins. Epitope mapping was performed forall mAbs using a panel of CCR5 alanine point mutants. Residues thataffected binding of all mAbs similarly were assumed to causeconformational perturbations in the co-receptor and not to constitutepart of the mAb epitopes. Only two such residues, Y10 and D11, have beenshown to affect HIV-1 entry (20, 52). The PA8, PA11 and PA12 epitopesare located exclusively in the Nt domain. Consistent with this result,PA8 was able to bind a biotinylated Nt peptide, containing residues D2through R31, in an ELISA (data not shown). However, PA11 and PA12, whosebinding strongly depended only on Q4, did not bind the Nt peptide insolution (data not shown). One possibility is that the Nt peptide doesnot assume the proper conformation for recognition by PA11 and PA12,whereas PA8 binding may be less conformation-dependent. Alternatively,PA11 and PA12 might interact with residues that we have not mutated, orform weak bonds with amino acids located in other domains of CCR5, orbind peptide backbone atoms whose presentation may be unchanged bymutagenesis. Antibodies PA9, PA10 and PA14 recognized epitopes thatincluded residues in both the Nt and ECL2 domains of CCR5, whereas the2D7 epitope was located exclusively in ECL2.

[0220] The PA14 epitope comprises both D2 in the Nt and R168 in ECL2indicating that these two residues are proximal to one another withinthe context of a mAb footprint. They may even directly interact with oneanother through their opposite charges.

[0221] MAbs PA8-PA12 and PA14 stained CCR5⁺ cells with differentintensities and in a cell type-dependent manner. All mAbs except PA8stained>90% L1.2-CCR5⁺ cells, the highest mean fluorescence intensitybeing observed with PA11 and PA12. However, PA14 and 2D7 stained thehighest percentage of PBMC and also yielded the highest meanfluorescence intensities on these cells. Hill et al. (28) have recentlycharacterized a panel of anti-CCR5 mAbs that similarly stainedtransfected cells, but only two of eight stained PBMC, and none stainedprimary monocytes. A low affinity for CCR5 probably accounted for thenon-reactivity of two of the mAbs with primary cells, but this wasunlikely to be the explanation for the failure of the other four toreact. In our mAb panel, we observe the most intense staining of PBMC bymAbs 2D7 and PA14 that have epitopes located entirely or partially inthe first ten residues of ECL2. Hill et al. report, however, that mAbsspecific for the Nt and ECL1 stain PBMCs, while mAbs to ECL2 and ECL3 donot stain PBMC, so a consistent pattern of reactivity has not beenidentified. One explanation for cell type-specific staining by mAbswould be that activated PBMCs (and monocytes) secrete CC-chemokines thatbind to cell surface CCR5, masking some mAb epitopes. However, one wouldexpect this to be especially true for PA14 and 2D7, which areantagonists of chemokine-induced calcium mobilization and presumablycompete with CC-chemokines for binding to CCR5. Yet these mAbs stainPBMC the most intensely. Alternatively, differential CCR5 epitopeexposure may reflect cell type-specific receptor oligomerization,association with other cell-surface molecules, or differentpost-translational modifications such as glycosylation. We have shownthat differences in mAb binding probably do not reflect cell typespecific differences in CD4/CCR5 interactions.

[0222] MAbs PA8-PA12 did not inhibit CC-chemokine induced calciummobilization in CCR5⁺ cells, nor did they mediate signaling throughCCR5. MAbs 2D7 and PA14 were inhibitors of CC-chemokine induced calciummobilization, but 2D7 was almost an order of magnitude more potent thanPA14. This may be because the PA14 epitope overlaps less with theCC-chemokine binding domain on CCR5 than the 2D7 epitope. All of themAbs also blocked HIV-1 entry and envelope-mediated membrane fusion, butinhibition of cell-cell fusion required in some cases almost two ordersof magnitude more antibody than what was needed to block viral entry.Presumably, more gp120/CD4/CCR5 interactions as well as interactionsbetween adhesion molecules are established and act cooperatively duringcell-cell fusion, compared to virus-cell fusion, making it moredifficult to inhibit. This is commonly observed with antibodies to LFA-1or to the HIV-1 envelope glycoprotein (45, 51). PA8, PA9 and PA10 wereunable to block cell-cell fusion by >15% and viral entry by >40%, evenat the highest antibody concentrations. However, >90% inhibition offusion could be attained with PA11, PA12 and PA14, and >90% inhibitionof entry could be attained with PA14. The most potent of the six mAbs inblocking fusion and entry was PA14, which was as effective as 2D7.Surprisingly, PA14 and 2D7 were among the least potent inhibitors ofgp120/sCD4 binding to L1.2-CCR5⁺ cells, whereas PA9-PA12 blocked withsimilar potencies, and PA8 was unable to block >40% of gp120/sCD4binding. These observations raise questions about the nature of the CCR5molecules presented on different cells and about the mechanisms ofinhibition of viral fusion and entry. It may be that CCR5 on L1.2 cells,used in the mAb and gp120-binding assays, is not in an identicalconformation to CCR5 on PBMC, used in the mAb-binding assay, or to CCR5on PM1 and U87MG cells used in the fusion and entry assays.

[0223] The low staining of PBMC and the partial inhibition of fusion andentry by some of our mAbs indicate that they are only able to bind to asubset of CCR5 molecules expressed on primary lymphocytes, PM1 andU87MG˜CD4⁺CCR5⁺ cell lines. Yet, other than PA8, all mAbs are able tostain >90% L1.2-CCR5⁺ cells and to completely block binding of thegp120/sCD4 complex to these cells. At least one difference betweenL1.2-CCR5⁺ and the other cells that we have used is the density ofco-receptor protein on the cell surface. Indeed, we estimate that theL1.2-CCR5⁺ cells express 10- to 100-fold more cell surface co-receptorthan PM1 and U87MG-CD4⁺CCR5⁺ cells. But when HeLa cells are engineeredto transiently express as much co-receptor as the L1.2-CCR5⁺ cell line,we are still unable to detect gp120/sCD4 binding to them (data notshown). Over-expression of CCR5 on L1.2, along with other cell-specificfactors therefore, might favor a co-receptor conformation thatprominently exposes the Nt, making it more accessible to both mAbs andgp120. Such a conformation might be induced by receptor oligomerization,by diminished or altered associations with cell surface proteins or byreceptor interactions with G proteins (25, 62). Do multipleconformations of CCR5 co-exist on the cell surface, and are they allpermissive for viral entry? The patterns of mAb reactivity would suggestso, since HIV-1 entry and fusion can occur, albeit at reduced levels, inthe presence of mAb concentrations that saturate epitopes required forgp120 binding to L1.2-CCR5+ cells. We favor the hypothesis that theco-receptor molecules present on L1.2-CCR5⁺ cells possess one HIV-1entry-competent conformation whereas CCR5 molecules on PBMC, PM1 andCCR5⁺ U87MG exist in multiple, entry-competent states that displaydifferent mAb reactivities. Whereas PA14 and 2D7 may recognize allconformations, other mAbs may not. Why L1.2 cells are conducive to aparticular fusion-competent conformation remains to be determined.

[0224] It has recently been demonstrated that the gp120-binding domainlies in the first twenty residues of the CCR5 Nt domain. MAbs to thegp120-binding domain on CCR5 potently block this interaction but are notnearly as efficient at inhibiting HIV-1 fusion and entry into targetcells as PA14 and 2D7, whose epitopes lie outside this region. PA14recognizes the tip of the Nt and residues in ECL2, whereas the 2D7epitope is located exclusively in ECL2. At the mechanism of action ofthese mAbs can only be speculated. It may be that their binding to thefirst few residues of ECL2 induces conformational changes in theco-receptor that prevent membrane fusion. Alternatively, obstruction ofECL2 epitopes might impede co-receptor oligomerization and the formationof a fusion-competent protein complex. Yet another possibility is thatresidues in ECL2 face the inside of the fusion pore and binding of themAbs impedes gp41 from inserting the fusion peptide into the plasmamembrane. In contrast, mAbs PA8-PA12 probably inhibit fusion and entryonly by directly competing for binding with gp120/CD4 complexes. We donot know if parameters other than epitope exposure and affinity for CCR5determine the efficacy of viral entry inhibition by these mAbs. It isunclear why inhibiting steps subsequent to the gp120/co-receptorinteraction would be more efficient than directly blocking thatinteraction. One way to explain this would be to assume that the offrate of gp120 binding to CCR5 is much lower than the on rate of mAbbinding to CCR5. Thus, every time a mAb detaches itself from aco-receptor molecule, a virion-associated gp120 molecule replaces it ina quasi-irreversible fashion since this interaction leads to membranefusion.

[0225] Synergy between combinations of anti-CCR5 mAbs is probably aresult of their interactions with distinct epitopes that are involved ininter-dependent, consecutive steps of HIV-1 entry. The degree of synergyobserved between PA12 and 2D7 (CI<0.1 under many circumstances) isextraordinary since CI values<0.2 are rarely observed for combinationsof anti-HIV-1 antibodies (33, 35, 61), reverse transcriptase inhibitors(29), or protease inhibitors (44). Because of its potency, the PA12:2D7combination was examined in multiple assay formats and concentrationratios, for which consistently high levels of synergy were observed.Moderate synergy was observed for PA12 combined with PA14. We alsoobserved moderate synergy between PA12 and CD4-IgG2. The CD4/gp120complex is metastable and if it is unable to interact with aco-receptor, decays into a non-fusogenic state (45-48). Since PA12directly blocks the gp120 -binding site on CCR5, its presence may shiftthe equilibrium towards inactivation of the gp120/CD4 complex. Thiswould explain why we observe synergy between CD4-IgG2 and mAb PA12 withrespect to inhibition of fusion and entry. The lack of synergy betweenmAb PA14 and CD4-IgG2 suggests that they act on two non-consecutive andindependent steps of viral entry. A combination of further studies willbe needed to determine the precise mechanisms of synergy of thedifferent compounds with respect to inhibition of viral fusion andentry.

[0226] The above results are consistent with a model wherein HIV-1 entryoccurs in three distinct steps involving receptor binding, co-receptorbinding, and co-receptor mediated membrane fusion. Separate co-receptorbinding and fusion events are suggested by the lack of correlationbetween the monoclonal antibodies' abilities to block gp120 binding andHIV-1 fusion/entry. The chronology of events during fusion is furthersuggested by the patterns of synergies observed. Agents, such as PA12,that potently inhibit the middle step of the process, namely gp 120binding, act synergistically with inhibitors of prior and subsequentsteps.

EXAMPLE 2

[0227] Background: The increasing incidence of multidrug-resistant HIV-1mandates the search for novel classes of antiretroviral agents. CCR5 isa requisite fusion coreceptor for primary HIV-1 isolates and provides apromising target for antiviral therapy. PRO140 is an anti-CCR5monoclonal antibody that potently inhibits HIV-1 entry and replicationat concentrations that do not affect CCR5's chemokine receptor activityin vitro. In the present study, we evaluated the therapeutic potentialof PRO 140 in vivo using a therapeutic animal model of HIV-1 infection.

[0228] Methods: CD-17 -SCID mice were reconstituted with normal humanPBMC and infected with the R5 isolate HIV-1 JR-CSF. When viral steadystate was reached, the animal were treated intraperitoneally with PRO140 or control antibody and monitored for viral burden using the RocheAmplicor assay. Initial studies examined a single 1 mg dose of PRO140.In multi-dose studies, PRO 140 was administered once every three daysfor three weeks at doses ranging from 0.1-1.0 mg. In a separateexperiment, flow cytometry was used to examine the potential forlymphocyte depletion following PRO 140 injection.

[0229] Results: Both single-dose and multi-dose PRO 140 reduced viralloads to undetectable levels in all treated animals, and the viral loadreductions ranged to 1.8 log 10. A transitory control of viralreplication was observed following single injection of PRO 140 whilemultiple injections led to a prolonged control with no evidence of viralrebound during therapy. Dose-dependent differences were observed in thekinetics of the PRO 140-mediated reductions in viral load. Flowcytometry analysis showed that treatment with PRO 140 did not lead tolymphocyte depletion, confirming that impact on viral replication invivo was solely due to CCR5-blockage.

[0230] Conclusions: PRO 140 is highly effective in controllingestablished HIV-1 infection in the hu-PBL-SCID mouse model of HIV-1infection. These findings provide in vivo proof-of-concept for PRO 140therapy in particular and for CCR5-inhibitors therapy in general.

EXAMPLE 3

[0231] Methods:

[0232] A humanized CCR5 antibody (huPRO 140) was tested for the abilityto block RANTES-induced calcium mobilization in L1.2-CCR5 cells and theability to block replication of HIV-1 CASE C 1/85 in human PBMC's usingmethods described herein.

[0233] Results:

[0234] The results as shown in FIG. 19 shows that the humanized CCR5antibody potently blocks HIV-1 but not RANTES.

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[0312]

1 13 1 31 PRT HUMAN 1 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp IleAsn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Lys Gln IleAla Ala Ala Arg 20 25 30 2 15 PRT HUMAN 2 His Tyr Ala Ala Ala Gln TrpAsp Phe Gly Asn Thr Met Cys Gln 1 5 10 15 3 27 PRT HUMAN 3 Arg Ser GlnLys Glu Gly Leu His Tyr Thr Cys Ser Ser His Phe Pro 1 5 10 15 Tyr SerGln Tyr Gln Phe Trp Lys Asn Phe Gln 20 25 4 17 PRT HUMAN 4 Gln Glu PhePhe Gly Leu Asn Asn Cys Ser Ser Ser Asn Arg Leu Asp 1 5 10 15 Gln 5 429DNA Homo sapiens 5 tctagaccac catgaagttg cctgttaggc tgttggtgctgatgttctgg attcctgctt 60 ccagcagtga tattgtgatg acccaatctc cactctccctgcctgtcact cctggagagc 120 cagcctccat ctcttgcaga tctagtcagc gccttctgagcagttatgga catacctatt 180 tacattggta cctacagaag ccaggccagt ctccacagctcctgatctac gaagtttcca 240 accgattttc tggggtccca gacaggttca gtggcagtgggtcagggaca gatttcacac 300 ttaagatcag tagagtggag gctgaggatg tgggagtttattactgctct caaagtacac 360 atgttcctct cacgttcgga caggggacca aggtggaaataaaacgtaag tagtcttctc 420 aactctaga 429 6 129 PRT Homo sapiens 6 Met LysLeu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 SerSer Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val 20 25 30 ThrPro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Leu Leu 35 40 45 SerSer Tyr Gly His Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 50 55 60 GlnSer Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly 65 70 75 80Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser 100 105110 Ser Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115120 125 Lys 7 393 DNA Homo sapiens 7 atgaagttgc ctgttaggct gttggtgctgatgttctgga ttcctgcttc cagcagtgat 60 attgtgatga cccaatctcc actctccctgcctgtcactc ctggagagcc agcctccatc 120 tcttgcagat ctagtcagcg ccttctgagcagttatggac atacctattt acattggtac 180 ctacagaagc caggccagtc tccacagctcctgatctacg aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgggtcagggacag atttcacact taagatcagt 300 agagtggagg ctgaggatgt gggagtttattactgctctc aaagtacaca tgttcctctc 360 acgttcggac aggggaccaa ggtggaaataaaa 393 8 457 DNA Homo sapiens 8 acgcgtccac catggaatgg agcggagtctttatctttct cctgtcagta actgcaggtg 60 tccactccga ggtgcagctg gtggagtctggtggaggctt ggtaaagcct ggaggttccc 120 ttagactctc ctgtgcagcc tctggttacactttcagtaa ctattggatc ggatgggtcc 180 gccaggctcc aggcaaaggg ctggagtggattggcgatat ctaccctgga gggaactaca 240 tcaggaacaa tgagaagttc aaggacaagaccaccctgtc agcagatact tccaagaaca 300 cagcctatct gcaaatgaac agcctgaaaaccgaggacac agccgtgtat tactgtggaa 360 gcagcttcgg tagtaactac gtgttcgcctggtttactta ctggggccaa gggactctgg 420 tcacagtctc ctcaggtgag tccttaaaacctctaga 457 9 141 PRT Homo sapiens 9 Met Glu Trp Ser Gly Val Phe Ile PheLeu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu ValGlu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Arg Leu SerCys Ala Ala Ser Gly Tyr Thr Phe 35 40 45 Ser Asn Tyr Trp Ile Gly Trp ValArg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Asp Ile Tyr ProGly Gly Asn Tyr Ile Arg Asn Asn 65 70 75 80 Glu Lys Phe Lys Asp Lys ThrThr Leu Ser Ala Asp Thr Ser Lys Asn 85 90 95 Thr Ala Tyr Leu Gln Met AsnSer Leu Lys Thr Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Gly Ser SerPhe Gly Ser Asn Tyr Val Phe Ala Trp Phe 115 120 125 Thr Tyr Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser 130 135 140 10 412 DNA Homo sapiens 10atggaatgga gcggagtctt tatctttctc ctgtcagtaa ctgcaggtgt ccactccgag 60gtgcagctgg tggagtctgg tggaggcttg gtaaagcctg gaggttccct tagactctcc 120tgtgcagcct ctggttacac tttcagtaac tattggatcg gatgggtccg ccaggctcca 180ggcaaagggc tggagtggat tggcgatatc taccctggag ggaactacat caggaacaat 240gagaagttca aggacaagac caccctgtca gcagatactt ccaagaacac agcctatctg 300caaatgaaca gcctgaaaac cgaggacaca gccgtgtatt actgtggaag cagcttcggt 360agtaactacg tgttcgcctg gtttacttac tggggccaag ggactctggt ca 412 11 457 DNAHomo sapiens 11 tctagaccac catggaatgg agcggggtct ttatctttct cctgtcagtaactgcaggtg 60 tccactccca ggtccacctg gtgcagtctg gacctgatgt gaaaaagcctgggacttcaa 120 tgaagatgtc ctgcaagacg tctggataca ccttcagtaa ctattggatcggatgggtta 180 ggcaggcgcc tggacaaggc cttgagtgga ttggagatat ttaccctggagggaactata 240 tcaggaacaa tgagaagttc aaggacaaga ccacactgac ggcagacacatcgaccagca 300 cggcctacat gcaacttggc agcctgagat ctgaagacac tgccgtctattactgtggaa 360 gcagcttcgg tagtaactac gtgttcgcct ggtttactta ctggggccaagggactctgg 420 tcacagtctc ctcaggtgag tccttaaaac ctctaga 457 12 141 PRTHomo sapiens 12 Met Glu Trp Ser Gly Val Phe Ile Phe Leu Leu Ser Val ThrAla Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Pro AspVal Lys Lys 20 25 30 Pro Gly Thr Ser Met Lys Met Ser Cys Lys Thr Ser GlyTyr Thr Phe 35 40 45 Ser Asn Tyr Trp Ile Gly Trp Val Arg Gln Ala Pro GlyGln Gly Leu 50 55 60 Glu Trp Ile Gly Asp Ile Tyr Pro Gly Gly Asn Tyr IleArg Asn Asn 65 70 75 80 Glu Lys Phe Lys Asp Lys Thr Thr Leu Thr Ala AspThr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Gln Leu Gly Ser Leu Arg Ser GluAsp Thr Ala Val 100 105 110 Tyr Tyr Cys Gly Ser Ser Phe Gly Ser Asn TyrVal Phe Ala Trp Phe 115 120 125 Thr Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser 130 135 140 13 423 DNA Homo sapiens 13 atggaatgga gcggggtctttatctttctc ctgtcagtaa ctgcaggtgt ccactcccag 60 gtccacctgg tgcagtctggacctgatgtg aaaaagcctg ggacttcaat gaagatgtcc 120 tgcaagacgt ctggatacaccttcagtaac tattggatcg gatgggttag gcaggcgcct 180 ggacaaggcc ttgagtggattggagatatt taccctggag ggaactatat caggaacaat 240 gagaagttca aggacaagaccacactgacg gcagacacat cgaccagcac ggcctacatg 300 caacttggca gcctgagatctgaagacact gccgtctatt actgtggaag cagcttcggt 360 agtaactacg tgttcgcctggtttacttac tggggccaag ggactctggt cacagtctcc 420 tca 423

What is claimed:
 1. An anti-CCR5 antibody which comprises (i) two lightchains, each light chain comprising the expression product of a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii)two heavy chains, each heavy chain comprising the expression product ofeither a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), or a fragment of suchantibody, which binds to CCR5 on the surface of a human cell.
 2. Theanti-CCR5 antibody of claim 1, wherein the heavy chains are expressed bythe plasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098).
 3. The anti-CCR5 antibody of claim 1, wherein the heavychains are expressed by the plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099).
 4. An anti-CCR5 antibodycomprising two light chains, each chain comprising consecutive aminoacids, the amino acid sequence of which is set forth in SEQ ID NO: 6,and two heavy chains, each heavy chain comprising consecutive aminoacids, the amino acid sequence of which is set forth in SEQ ID NO:
 9. 5.An anti-CCR5 antibody comprising two light chains, each light chaincomprising consecutive amino acids, the amino acid sequence of which isset forth in SEQ ID NO: 6, and two heavy chains, each heavy chaincomprising consecutive amino acids, the amino acid sequence of which isset forth in SEQ ID NO:
 12. 6. An isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO:
 6. 7. The nucleic acid of claim 6,wherein the consecutive amino acids are the amino acids expressed by aplasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097).8. The nucleic acid of claim 6, wherein the nucleic acid comprises thesequence set forth in SEQ ID NO:
 5. 9. The nucleic acid of any one ofclaims 6, 7 or 8, wherein the nucleic acid is RNA, DNA or cDNA.
 10. Anisolated nucleic acid encoding a polypeptide comprising consecutiveamino acids, the amino acid sequence of which is set forth in SEQ ID NO:9.
 11. The nucleic acid of claim 10, wherein the consecutive amino acidsare the amino acids expressed by a plasmid designated pVg1:HuPRO140HG2-VH (ATCC Deposit Designation PTA-4098).
 12. The nucleic acid ofclaim 10, wherein the nucleic acid comprises the sequence set forth inSEQ ID NO:
 8. 13. The nucleic acid of any one of claims 10, 11 or 12wherein the nucleic acid is RNA, DNA or cDNA.
 14. An isolated nucleicacid encoding a polypeptide comprising consecutive amino acids, theamino acid sequence of which is set forth in SEQ ID NO:
 12. 15. Thenucleic acid of claim 14, wherein the consecutive amino acids are theamino acids expressed by a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099)
 16. The nucleic acid ofclaim 14, wherein the nucleic acid comprises the sequence set forth inSEQ ID NO:
 11. 17. The nucleic acid of any one of claims 14, 15 and 16,wherein the nucleic acid is RNA, DNA or cDNA.
 18. A compositioncomprising at least one of the anti-CCR5 antibody or a fragment thereof,of any one of claims 1-5 and a carrier.
 19. A composition comprising theanti-CCR5 antibody or a fragment thereof, of any one of claims 1-5,having attached thereto a material selected from the group consisting ofa radioisotope, a toxin, polyethylene glycol, a cytotoxic agent and adetectable label.
 20. A method of inhibiting HIV-1 infection of a CD4+cell which comprises contacting the CD4+ cell with an antibody whichcomprises (i) two light chains, each light chain comprising theexpression product of a plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097), and (ii) two heavy chains, each heavy chaincomprising the expression product of either a plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099), or a fragment of such antibody which binds to CCR5 on thesurface of the CD4+ cell, in an amount and under conditions such thatfusion of HIV-1 or an HIV-1 infected cell to-the CD4+ cell is inhibited,thereby inhibiting HIV-1 infection of the CD4+ cell.
 21. The method ofclaim 20, wherein the CD4+ cell expresses CCR5.
 22. A method of treatinga subject afflicted with HIV-1 which comprises administering to thesubject an effective HIV-1 treating dosage amount of an anti-CCR5antibody comprising (i) two light chains, each light chain comprisingthe expression product of a plasmid designated pVK:HuPRO140-VK (ATCCDeposit Designation PTA-4097), and (ii) two heavy chains, each heavychain comprising the expression product of either a plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099), or a fragment of such antibody, which binds to CCR5 on thesurface of a human cell, under conditions effective to treat saidHIV-1-afflicted subject.
 23. A method of preventing a subject fromcontracting an HIV-1 infection which comprises administering to thesubject an effective HIV-1 infection-preventing dosage amount of ananti-CCR5 antibody comprising (i) two light chains, each light chaincomprising the expression product of a plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavychains, each heavy chain comprising the expression product of either aplasmid designated pVg1:HuPRO 140 HG2-VH (ATCC Deposit DesignationPTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCCDeposit Designation PTA-4099), or a fragment of such antibody, whichbinds to CCR5 on the surface of a human cell, under conditions effectiveto prevent said HIV-1 infection in said subject.
 24. The method of claim22 or 23, wherein the anti-CCR5 antibody is administered to the subjectby a method selected from the group consisting of intravenous,intramuscular and subcutaneous means.
 25. The method of claim 22 or 23,wherein the anti-CCR5 antibody is administered continuously to saidsubject.
 26. The method of claim 22 or 23 wherein the anti-CCR5 antibodyis administered at predetermined periodic intervals to said subject. 27.The method of claim 22 or 23, which further comprises labeling theanti-CCR5 antibody with a detectable marker.
 28. The method of claim 27,wherein the detectable marker is a radioactive or a fluorescent marker.29. The method of claim 22 or 23, wherein the dosage of said anti-CCR5antibody ranges from about 0.1 to about 100,000 μg/kg body weight ofsaid subject.
 30. The method of claim 29, wherein the dosage of saidanti-CCR5 antibody does not inhibit an endogenous chemokine activity onCCR5 in said subject.
 31. An anti-CCR5 antibody conjugate comprising ananti-CCR5 antibody which comprises (i) two light chains, each lightchain comprising the expression product of a plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavychains, each heavy chain comprising the expression product of either aplasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCCDeposit Designation PTA-4099), or a fragment of such antibody whichbinds to CCR5 on the surface of a human cell, conjugated to at least onepolymer.
 32. The anti-CCR5 antibody conjugate of claim 31, wherein thepolymer is selected from the group consisting of hydrophilic polyvinylpolymers, polyalkylene ethers, polyoxyalkylenes, polymethacrylates,carbomers, branched polysaccharides, unbranched polysaccharides,polymers of sugar alcohols, heparin and heparon.
 33. The anti-CCR5antibody conjugate of claim 32, wherein the polyalkylene ether ispolyethylene glycol (PEG) or a derivative thereof.
 34. The anti-CCR5antibody conjugate of claim 33, wherein at least one PEG has an averagemolecular weight of at least 20 kD.
 35. The anti-CCR5 antibody conjugateof claim 31, wherein the apparent size of the conjugate is at leastabout 500 kD.
 36. The anti-CCR5 antibody conjugate of claim 31, whereinthe conjugate has at least one of an increase in serum half-life, anincrease in mean residence time in the circulation and a decrease inserum clearance rate, compared to a nonconjugated anti-CCR5 antibody orfragment thereof.
 37. A method of inhibiting infection of a CCR5+ cellby HIV-1, which method comprises administering to a subject at risk ofHIV-1 infection the conjugate of claim 31 in an amount and underconditions effective to inhibit infection of CCR5+ cells of said subjectby HIV-1.
 38. A method of treating an HIV-1 infection in a subject,which method comprises administering to an HIV-1-infected subject theconjugate of claim 31 in an amount and under conditions effective totreat the subject's HIV-1 infection.
 39. The method of claim 38, whereinthe amount of the conjugate is effective in reducing a viral load in thesubject.
 40. The method of claim 38, wherein the amount of the conjugateis effective in increasing a CD4+ cell count in the subject.
 41. Themethod of claim 38, which further comprises administering to saidsubject at least one conventional anti-viral agent.
 42. The method ofclaim 37 or 38, wherein the conjugate is administered to the subject bya method selected from the group consisting of intravenous,intramuscular and subcutaneous means.
 43. The method of claim 37 or 38,wherein the conjugate is administered continuously to said subject. 44.The method of claim 37 or 38, wherein the conjugate is administered atpredetermined periodic intervals to said subject.
 45. The method ofclaim 37 or 38, which further comprises labeling the conjugate with adetectable marker.
 46. The method of claim 45, wherein the detectablemarker is a radioactive or a fluorescent marker.
 47. A transformed hostcell comprising at least two vectors, at least one vector comprising anucleic acid sequence encoding heavy chains of an anti-CCR5 antibody,and at least one vector comprising a nucleic acid sequence encodinglight chains of the anti-CCR5 antibody, wherein the anti-CCR5 antibodycomprises two heavy chains having the amino acid sequence set forth inSEQ ID NO: 9, and two light chains having the amino acid sequence setforth in SEQ ID NO:
 6. 48. A transformed host cell comprising at leasttwo vectors, at least one vector comprising a nucleic acid sequenceencoding heavy chains of an anti-CCR5 antibody, and at least one vectorcomprising a nucleic acid sequence encoding light chains of theanti-CCR5 antibody, wherein the anti-CCR5 antibody comprises two heavychains having the amino acid sequence set forth in SEQ ID NO: 12, andtwo light chains having the amino acid sequence set forth in SEQ ID NO:6.
 49. The transformed host cell of claim 47 or 48, wherein the cell isa mammalian cell.
 50. The transformed host cell of claim 49 wherein thecell is a COS cell, a CHO cell or a myeloma cell.
 51. The transformedhost cell of claim 47 or 48, wherein the cell secretes the anti-CCR5antibody.
 52. The transformed host cell of claim 47, wherein the vectorencoding heavy chains is designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098).
 53. The transformed host cell of claim 48,wherein the vector encoding heavy chains is designated pVg1:HuPRO140(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099).
 54. The transformedhost cell of claim 47 or 48, wherein the vector encoding light chains isdesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097).
 55. Thetransformed host cell of claim 47, wherein the vector encoding heavychains is designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098) and the vector encoding light chains is designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097).
 56. The transformedhost cell of claim 48, wherein the vector encoding the heavy chains isdesignated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099) and the vector encoding light chains is designatedpVK-HuPRO140-VK (ATCC Deposit Designation PTA-4097).
 57. The transformedhost cell of claim 47, wherein the nucleic acid sequence encoding heavychains has the nucleic acid sequence set forth in SEQ. ID NO:
 8. 58. Thetransformed host cell of claim 48, wherein the nucleic acid sequenceencoding heavy chains has the nucleic acid sequence set forth in SEQ IDNO:
 11. 59. The transformed host cell of claim 47 or 48 wherein thenucleic acid sequence encoding light chains has the nucleic acidsequence set forth in SEQ ID NO:
 5. 60. A vector comprising a nucleicacid sequence encoding a heavy chain of an anti-CCR5 antibody, whereinthe heavy chain comprises the amino acid sequence set forth in SEQ IDNO:
 9. 61. The vector of claim 60, wherein the vector is designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation No. PTA-4098).
 62. Avector comprising a nucleic acid sequence encoding a heavy chain of ananti-CCR5 antibody, wherein the heavy chain comprises the amino acidsequence set forth in SEQ ID NO:
 12. 63. The vector of claim 62, whereinthe vector is designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC DepositDesignation No. PTA-4099).
 64. A vector comprising a nucleic acidsequence encoding a light chain of an anti-CCR5 antibody, wherein thelight chain comprises the amino acid sequence set forth in SEQ ID NO: 6.65. The vector of claim 64, wherein the vector is designatedpVK:HuPRO140-VK (ATCC Deposit Designation No. PTA-4097).
 66. A processfor producing an anti-CCR5 antibody which comprises culturing a hostcell containing therein (i) a plasmid designated pVK:HuPRO140-VK (ATCCDeposit Designation PTA-4097), and (ii) either a plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg1:PRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099) under conditions permitting the production of an antibodycomprising two light chains encoded by the plasmid designatedpVK:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4097)and two heavychains encoded either by the plasmid designated pVg1:HuPRO140 HG2-VH(ATCC Deposit Designation PTA-4098) or by the plasmid designatedpVg1:HuPRO 140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), so asto thereby produce an anti-CCR5 antibody.
 67. A process for producing ananti-CCR5 antibody which comprises: a) transforming a host cell with (i)a plasmid designated pVK:HuPRO140-VK (ATCC Deposit DesignationPTA-4097), and (ii) either a plasmid designated pVg1:HuPRO140 HG2-VH(ATCC Deposit Designation PTA-4098) or a plasmid designatedpVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099); and b)culturing the transformed host cell under conditions permittingproduction of an antibody comprising two light chains encoded by theplasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097)and two heavy chains encoded either by the plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or by theplasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC DepositDesignation PTA-4099), so as to thereby produce an anti-CCR5 antibody.68. The method of claim 66 or 67, which further comprises recovering theanti-CCR5 antibody so produced in isolated form.
 69. The method of claim66 or 67, wherein the host cell is a mammalian cell.
 70. The method ofclaim 69, wherein the mammalian host cell is a COS cell, a CHO cell or amyeloma cell.
 71. The method of claim 66 or 67, wherein the heavy chainsof the anti-CCR5 antibody are encoded by the plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098).
 72. The methodof claim 66 or 67, wherein the heavy chains of the anti-CCR5 antibodyare encoded by the plasmid designated pVg1:HuPRO140 (mut B+D+I) (ATCCDeposit Designation PTA-4099).
 73. A kit for use in a process ofproducing an anti-CCR5 antibody comprising: a) a vector comprising anucleic acid sequence encoding a light chain of an anti-CCR5 antibody,wherein the light chain comprises the amino acid sequence set forth inSEQ ID NO: 6; and b) a vector comprising a nucleic acid sequenceencoding a heavy chain of an anti-CCR5 antibody, wherein the heavy chaincomprises the amino acid sequence set forth in SEQ ID NO: 9, or a vectorcomprising a nucleic acid sequence encoding a heavy chain of ananti-CCR5 antibody, wherein the heavy chain comprises the amino acidsequence set forth in SEQ ID NO: 12.